PI-3 kinase inhibitor prodrugs

ABSTRACT

The invention provides novel prodrugs of inhibitors of PI-3 kinase. The novel compounds are LY294002 and analogs thereof comprising a reversibly quaternized amine.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/111,201, filed Apr. 20, 2005, now U.S. Pat. No. 7,396,828 which is acontinuation of U.S. application Ser. No. 10/818,145, filed Apr. 5,2004, which issued as U.S. Pat. No. 6,949,537; and which claims thebenefit of U.S. Provisional Application No. 60/460,137, filed Apr. 3,2003, the contents of each which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to prodrugs of PI-Kinase inhibitors andmethods of using these inhibitors.

2. Description of Related Art

PI-3 kinases are a large family of lipid kinases that phosphorylatephosphatidylinositol in the D3 position to generate an important secondmessenger, phosphatidylinositol 3′-phosphate. Members of the PI-3 kinasefamily are divided into 3 classes based on sequence homology and theproduct formed by enzyme catalysis. The class I PI-3 kinases arecomposed of 2 subunits: a 110 kd catalytic subunit and an 85 kdregulatory subunit. Class I PI-3 kinases are involved in importantsignal transduction events downstream of cytokines, integrins, growthfactors and immunoreceptors, which suggests that control of this pathwaymay lead to important therapeutic effects.

Inhibition of class I PI-3 kinase induces apoptosis, blocks tumorinduced angiogenesis in vivo, and increases the radiosensitivity ofcertain tumors. LY294002(2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) (Compound 1) is awell known specific inhibitor of class I PI-3 kinases and has beendemonstrated to possess anti-cancer properties.

However, the anti-cancer applications of LY294002 are severely limitedby its lack of aqueous solubility and its poor pharmacokinetics.Moreover, LY294002 has no tissue specific properties and has beendemonstrated to be rapidly metabolized in animals. Because of thesefactors, LY294002 would need to be administered at frequent intervalsand thus has the potential to also inhibit PI-3 kinases in normal cellsthereby leading to undesirable side effects.

There continues to be a need for class I PI-3 kinase inhibitors withimproved pharmacokinetic and pharmacodynamic properties. The presentinvention fulfills these needs and provides other related advantages.

SUMMARY OF THE INVENTION

The present invention is related to pro-compounds comprising aquaternary nitrogen, wherein one bond of the quaternary nitrogen ishydrolyzable and after hydrolysis of said bond yields a compound of theformula:

wherein,

-   -   R₁ and R₂ independently are H, optionally substituted aliphatic,        optionally substituted aryl, hydroxyl, halogen, alkoxy,        heterocycle, cyano, amino, or, are taken together to form an        optionally substituted cycloaliphatic or optionally substituted        aryl;    -   R₃ represents H, optionally substituted aliphatic, and        optionally substituted aryl; and    -   R₄ and R₅ independently are H, optionally substituted aliphatic,        optionally substituted aryl, heterocycle, aryloxy, carboxy, or,        are taken together to form an optionally substituted heterocycle        or optionally substituted heteroaryl.

The pro-compound may be of the formula:

wherein,

-   -   Ring A is benzo;    -   Z₁ and Z₂ independently are S or O;    -   R₁ and R₂ independently are H, optionally substituted aliphatic,        optionally substituted aryl, hydroxyl, halogen, alkoxy,        heterocycle, cyano, amino, or, are taken together to form an        optionally substituted cycloaliphatic or optionally substituted        aryl;    -   R₃ represents H, optionally substituted aliphatic, and        optionally substituted aryl;    -   R₄ and R₅ independently are H, optionally substituted aliphatic,        optionally substituted aryl, heterocycle, aryloxy, carboxy, or,        are taken together to form an optionally substituted heterocycle        or optionally substituted heteroaryl;    -   R₆ represents H, optionally substituted aliphatic, optionally        substituted aryl, alkoxy, carboxy, amino, heterocycle, aryloxy,        and optionally substituted therewith a targeting agent; and    -   L represent a linker group.        The targeting agent may be a vitamin, peptide, protein,        liposome, bone-seeking agent or cartilage-seeking agent

The present invention is also related to intermediate compounds of theformula:

wherein,

-   -   X represents a halo group, preferably Cl or I;    -   Y represents —CH₂—, —CH(CH₃)—, —CH(Ph)-, —C(CH3)(COOH)— or        CH(CH(CH3)2)—;    -   Z₁ and Z₂ independently are S or O; and    -   n=0 to 1.

The present invention is also related to the use of the pro-compoundsfor the treatment of a condition associated with PI-3 kinase activity,inflammatory disease, age-related macular degeneration, conditionsassociates with a mutant PTEN, hypertension, pancreatitis, ulcers,cancer; disrupting leukocyte function; inducing apoptosis; enhancing thechemosensitivity of tumor cells; enhancing the radiosensitivity of tumorcells; inhibiting tumor induced angiogenesis; inhibiting angiogenicprocesses associated with non-cancer diseases; improving performance ofa stent; inhibiting phosphatidylinositol 3-kinase in a whole cell of apatient, comprising administering to a patient in need thereof aneffective amount of a composition comprising the pro-compounds of thepresent invention. The pro-compounds may be administered to the patientby slow I.V. fusion.

The present invention is also related to a method for suppressingdifferentiation of progenitor cells comprising contacting progenitorcells with an effective amount of a composition comprising apro-compound of the present invention.

The present invention is also related to the purification of thepro-compounds of the present invention comprising adding thepro-compounds to a solution comprising at least 0.1% (v/v) acid. Thesolution comprising the pro-compound is the chromatographed, preferablyby HPLC, to isolate the pro-compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structure of EDTMP, DOTMP, ABDTMP, BAD, MTX-BPand CF—BP.

FIG. 2 shows the chemical structures of potential bone targeting agents.

FIG. 3 shows the chemical reaction for modifying a phosphonate in a bonetargeting agent.

FIG. 4 shows the alkylation reaction to modify a phosphonate in a bonetargeting agent.

FIG. 5 shows a concept for chemically modifying EDTMP and DOTMP.

FIG. 6 shows the inhibition of phagocytosis by LY294002 in J774 cells.The columns indicate phagocytic index or percentage of cells positivefor phagocytic response. The phagocytic index is the number of sRBC's(sheep red blood cells) found per 100 J774 cells and the % of phagocyticcells is the % of J774 cells that have phagocytized at least 1 sRBC. Theerror bars represent standard deviation of mean.

FIG. 7 shows the UV and ELS Chromatograms of Compound 1126 (AO36-33).

FIG. 8 shows the Positive Mass Spectrum of Compound 1126 (A036-33).

FIG. 9 shows that Avβ3 targeted PI 3 kinase inhibitors abrogated thetube formation of EDC-CBF1 endothelial cells on Matrigel.

DETAILED DESCRIPTION

Before the present compounds, products and compositions and methods aredisclosed and described, it is to be understood that the terminologyused herein is for the purpose of describing particular embodiments onlyand is not intended to be limiting. It must be noted that, as used inthe specification and the appended claims, the singular forms “a,” “an”and “the” include plural referents unless the context clearly dictatesotherwise.

Throughout this application, where publications are referenced, thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

1. DEFINITIONS

The term “branched” as used herein refers to a group containing from 1to 24 backbone atoms wherein the backbone chain of the group containsone or more subordinate branches from the main chain. Preferred branchedgroups herein contain from 1 to 12 backbone atoms. Examples of branchedgroups include, but are not limited to, isobutyl, t-butyl, isopropyl,—CH₂CH₂CH(CH3)CH₂CH₃, —CH₂CH(CH₂CH₃)CH₂CH₃, —CH₂CH₂C(CH₃)₂CH₃,—CH₂CH₂C(CH₃)₃ and the like.

The term “unbranched” as used herein refers to a group containing from 1to 24 backbone atoms wherein the backbone chain of the group extends ina direct line. Preferred unbranched groups herein contain from 1 to 12backbone atoms.

The term “cyclic” or “cyclo” as used herein alone or in combinationrefers to a group having one or more closed rings, whether unsaturatedor saturated, possessing rings of from 3 to 12 backbone atoms,preferably 3 to 7 backbone atoms.

The term “lower” as used herein refers to a group with 1 to 6 backboneatoms.

The term “saturated” as used herein refers to a group where allavailable valence bonds of the backbone atoms are attached to otheratoms. Representative examples of saturated groups include, but are notlimited to, butyl, cyclohexyl, piperidine and the like.

The term “unsaturated” as used herein refers to a group where at leastone available valence bond of two adjacent backbone atoms is notattached to other atoms. Representative examples of unsaturated groupsinclude, but are not limited to, —CH₂CH₂CH═CH₂, phenyl, pyrrole and thelike.

The term “aliphatic” as used herein refers to an unbranched, branched orcyclic hydrocarbon group, which may be substituted or unsubstituted, andwhich may be saturated or unsaturated, but which is not aromatic. Theterm aliphatic further includes aliphatic groups, which comprise oxygen,nitrogen, sulfur or phosphorous atoms replacing one or more carbons ofthe hydrocarbon backbone.

The term “aromatic” as used herein refers to an unsaturated cyclichydrocarbon group having 4n+2 delocalized π(pi) electrons, which may besubstituted or unsubstituted. The term aromatic further includesaromatic groups, which comprise a nitrogen atom replacing one or morecarbons of the hydrocarbon backbone. Examples of aromatic groupsinclude, but are not limited to, phenyl, naphthyl, thienyl, furanyl,pyridinyl, (is)oxazoyl and the like.

The term “substituted” as used herein refers to a group having one ormore hydrogens or other atoms removed from a carbon or suitableheteroatom and replaced with a further group. Preferred substitutedgroups herein are substituted with one to five, most preferably one tothree substituents. An atom with two substituents is denoted with “di,”whereas an atom with more than two substituents is denoted by “poly.”Representative examples of such substituents include, but are notlimited to aliphatic groups, aromatic groups, alkyl, alkenyl, alkynyl,aryl, alkoxy, halo, aryloxy, carbonyl, acryl, cyano, amino, nitro,phosphate-containing groups, sulfur-containing groups, hydroxyl,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, acylamino, amidino, imino, alkylthio, arylthio,thiocarboxylate, alkylsulfinyl, trifluoromethyl, azido, heterocyclyl,alkylaryl, heteroaryl, semicarbazido, thiosemicarbazido, maleimido,oximino, imidate, cycloalkyl, cycloalkylcarbonyl, dialkylamino,arylcycloalkyl, arylcarbonyl, arylalkylcarbonyl, arylcycloalkylcarbonyl,arylphosphinyl, arylalkylphosphinyl, arylcycloalkylphosphinyl,arylphosphonyl, arylalkylphosphonyl, arylcycloalkylphosphonyl,arylsulfonyl, arylalkylsulfonyl, arylcycloalkylsulfonyl, combinationsthereof, and substitutions thereto.

The term “unsubstituted” as used herein refers to a group that does nothave any further groups attached thereto or substituted therefor.

The term “alkyl” as used herein alone or in combination refers to abranched or unbranched, saturated aliphatic group. Representativeexamples of alkyl groups include, but are not limited to, methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, octyl,decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like.

The term “alkenyl” as used herein alone or in combination refers to abranched or unbranched, unsaturated aliphatic group containing at leastone carbon-carbon double bond which may occur at any stable point alongthe chain. Representative examples of alkenyl groups include, but arenot limited to, ethenyl, E- and Z-pentenyl, decenyl and the like.

The term “alkynyl” as used herein alone or in combination refers to abranched or unbranched, unsaturated aliphatic group containing at leastone carbon-carbon triple bond which may occur at any stable point alongthe chain. Representative examples of alkynyl groups include, but arenot limited to, ethynyl, propynyl, propargyl, butynyl, hexynyl, decynyland the like.

The term “aryl” as used herein alone or in combination refers to asubstituted or unsubstituted aromatic group, which may be optionallyfused to other aromatic or non-aromatic cyclic groups. Representativeexamples of aryl groups include, but are not limited to, phenyl, benzyl,naphthyl, benzylidine, xylyl, styrene, styryl, phenethyl, phenylene,benzenetriyl and the like.

The term “alkoxy” as used herein alone or in combination refers to analkyl, alkenyl or alkynyl group bound through a single terminal etherlinkage. Examples of alkoxy groups include, but are not limited to,methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, 2-butoxy,tert-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy, neopentoxy,n-hexoxy, 2-hexoxy, 3-hexoxy, 3-methylpentoxy, fluoromethoxy,difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, andtrichloromethoxy.

The term “aryloxy” as used herein alone or in combination refers to anaryl group bound through a single terminal ether linkage.

The term “halogen,” “halide” or “halo” as used herein alone or incombination refers to fluorine “F”, chlorine “Cl”, bromine “Br”, iodine“I”, and astatine “At”. Representative examples of halo groups include,but are not limited to, chloroacetamido, bromoacetamido, idoacetamidoand the like.

The term “hetero” as used herein combination refers to a group thatincludes one or more atoms of any element other than carbon or hydrogen.Representative examples of hetero groups include, but are not limitedto, those groups that contain heteroatoms including, but not limited to,nitrogen, oxygen, sulfur and phosphorus.

The term “heterocycle” as used herein refers to a cyclic groupcontaining a heteroatom. Representative examples of heterocyclesinclude, but are not limited to, pyridine, piperadine, pyrimidine,pyridazine, piperazine, pyrrole, pyrrolidinone, pyrrolidine, morpholine,thiomorpholine, indole, isoindole, imidazole, triazole, tetrazole,furan, benzofuran, dibenzofuran, thiophene, thiazole, benzothiazole,benzoxazole, benzothiophene, quinoline, isoquinoline, azapine,naphthopyran, furanobenzopyranone and the like.

The term “carbonyl” or “carboxy” as used herein alone or in combinationrefers to a group that contains a carbon-oxygen double bond.Representative examples of groups which contain a carbonyl include, butare not limited to, aldehydes (i.e., formyls), ketones (i.e., acyls),carboxylic acids (i.e., carboxyls), amides (i.e., amidos), imides (i.e.,imidos), esters, anhydrides and the like.

The term “acryl” as used herein alone or in combination refers to agroup represented by CH₂═C(Q)C(O)O— where Q is an aliphatic or aromaticgroup.

The term “cyano,” “cyanate,” or “cyanide” as used herein alone or incombination refers to a carbon-nitrogen double bond. Representativeexamples of cyano groups include, but are not limited to, isocyanate,isothiocyanate and the like.

The term “amino” as used herein alone or in combination refers to agroup containing a backbone nitrogen atom. Representative examples ofamino groups include, but are not limited to, alkylamino, dialkylamino,arylamino, diarylamino, alkylarylamino, alkylcarbonylamino,arylcarbonylamino, carbamoyl, ureido and the like.

The term “phosphate-containing group” as used herein refers to a groupcontaining at least one phosphorous atom in an oxidized state.Representative examples include, but are not limited to, phosphonicacids, phosphinic acids, phosphate esters, phosphinidenes, phosphinos,phosphinyls, phosphinylidenes, phosphos, phosphonos, phosphoranyls,phosphoranylidenes, phosphorosos and the like.

The term “sulfur-containing group” as used herein refers to a groupcontaining a sulfur atom. Representative examples include, but are notlimited to, sulfhydryls, sulfenos, sulfinos, sulfinyls, sulfos,sulfonyls, thios, thioxos and the like.

The term “optional” or “optionally” as used herein means that thesubsequently described event or circumstance may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not. For example, the phrase“optionally substituted alkyl” means that the alkyl group may or may notbe substituted and that the description includes both unsubstitutedalkyl and alkyl where there is a substitution.

The term “effective amount,” when used in reference to a compound,product, or composition as provided herein, means a sufficient amount ofthe compound, product or composition to provide the desired result. Theexact amount required will vary depending on the particular compound,product or composition used, its mode of administration and the like.Thus, it is not always possible to specify an exact “effective amount.”However, an appropriate effective amount may be determined by one ofordinary skill in the art informed by the instant disclosure using onlyroutine experimentation.

The term “suitable” as used herein refers to a group that is compatiblewith the compounds, products, or compositions as provided herein for thestated purpose. Suitability for the stated purpose may be determined byone of ordinary skill in the art using only routine experimentation.

The term “hydrolyzable” as used herein refers to whether the group iscapable of or prone to hydrolysis (i.e., splitting of the molecule orgroup into two or more new molecules or group).

2. COMPOUNDS

The present invention provides a compound, which upon cleavage of onebond of a quaternary amine yields a compound of the formula:

wherein,

-   -   Ring A is benzo;    -   Z₁ and Z₂ independently are S or O;    -   R₁ and R₂ independently are H, optionally substituted aliphatic,        optionally substituted aryl, hydroxyl, halogen, alkoxy,        heterocycle, cyano, amino, or, are taken together to form an        optionally substituted cycloaliphatic or optionally substituted        aryl;    -   R₃ represents H, optionally substituted aliphatic, and        optionally substituted aryl; and    -   R₄ and R₅ independently are H, optionally substituted aliphatic,        optionally substituted aryl, heterocycle, aryloxy, carboxy, or,        are taken together to form an optionally substituted heterocycle        or optionally substituted heteroaryl.        Preferably, cleavage of one bond of a quaternary amine yields        LY294002 (Compound 1).

The present invention also provides a compound of the formula:

wherein,

-   -   Ring A is benzo;    -   Z₁ and Z₂ independently are S or O;    -   R₁ and R₂ independently are H, optionally substituted aliphatic,        optionally substituted aryl, hydroxyl, halogen, alkoxy,        heterocycle, cyano, amino, or, are taken together to form an        optionally substituted cycloaliphatic or optionally substituted        aryl;    -   R₃ represents H, optionally substituted aliphatic, and        optionally substituted aryl;    -   R₄ and R₅ independently are H, optionally substituted aliphatic,        optionally substituted aryl, heterocycle, aryloxy, carboxy, or,        are taken together to form an optionally substituted heterocycle        or optionally substituted heteroaryl; and    -   R₆ represents H, optionally substituted aliphatic, optionally        substituted aryl, alkoxy, carboxy, amino, heterocycle, aryloxy,        and optionally substituted therewith a targeting agent; and    -   L represents a linker group.

In a preferred embodiment, Compounds 2-3 of the present invention arethose compounds wherein, R₁-Ring A-R₂ is selected from the groupconsisting of the following:

In a preferred embodiment, Compounds 2-3 of the present invention arethose compounds wherein, R₄—N—R₅ is selected from the group consistingof the following:

In another embodiment Compounds 2-3 of the present invention are thosecompounds wherein, R₆ is selected from the group consisting of thefollowing:

a. Linker

In another embodiment, Compound 3 of the present invention are thosecompounds wherein, the linker group is hydrolyzable. The linker group ofthe prodrug may be cleaved by enzymatic cleavage or preferably byhydrolysis under physiological conditions including but not limited to,aqueous conditions in living animals, to yield Compound 2. The rate ofhydrolysis of the linker group under physiological conditions ispreferably from about 1 minute half-life to about 48 hour half-life.

b. Hydrolysis

The term “hydrolyzable” as used herein refers to whether the group iscapable of or prone to hydrolysis (i.e., splitting of the molecule orgroup into two or more new molecules or groups due to the net insertionof a water molecule) at a rate of about 1 minute half-life to 48 hourhalf-life.

The linker group may be any group that may be hydrolyzed orenzymatically cleaved to yield Compound 2. In a preferred embodiment,the linker group is of the formula:

wherein,

-   -   Z₃ and Z₄ independently are S or O; and    -   R₇ represents —CH₂—, —CH(CH₃)—, —CH(Ph)-, —C(CH3)(COOH)— or        CH(CH(CH3)2)—.

A representative example of the hydrolysis of the linker group of theprodrug to yield Compound 2 is presented in (Scheme 1), wherein Z₃ andZ₄ independently are each O. Hydrolysis or enzymatic cleavage of the R₆ester yields a hemiaminal that collapses with liberation of the R₇aldehyde, thereby generating Compound 2 comprising a free tertiaryamine. Both R₆ and R₇ can be selected to give different rates ofconversion back to the free tertiary amine. For example, increasingsubstitution at R₆ or R₇, or a combination thereof, may increase thestability towards hydrolysis. Furthermore, electron withdrawing groupson the R₆ moiety decreases the stability. In addition to the varying ofR₆ or R₇ disclosed herein, additional factors that may be vary thestability of the quaternary amine may be found in N. Bodor, Journal ofMedicinal Chemistry 1980, vol 23 #5 pp 469-480 “Soft Drugs. 1. LabileQuaternary Ammonium Salts as Soft Antimicrobials”; and G. Brouillette etal; Journal of Pharmaceutical Sciences, 1996, vol 85 #6, pp 620-623, thecontents of which are incorporated herein by reference.

c. Targeting Agent

In another embodiment, compounds of the present invention are thosecompounds wherein, R₆ further comprises one or more targeting agents (T)covalently attached thereto. Targeting agents allow the prodrugs of thepresent invention to be delivered selectively to specific types ofcells, tissues, organs or extracellular structures. As discussed above,treatment with Compound 1 (LY294002) suffers from poor bioavailability,rapid metabolism and side effects because the compound is not tissuespecific. Therefore, it is highly desirable to limit the location of thedrug to that of the area of treatment or at least prevent it fromreaching the tissues where if can cause side effects, and to ensure thatat any particular time effective, but not excessive, amounts of the drugare used. The use of targeting agents may allow the prodrugs of thepresent invention to be concentrated at the site of treatment ratherthan evenly distributed throughout the entire body or to be metabolizedprematurely or excreted too quickly. Once being delivered to the site oftreatment, the linker may be enzymatically cleaved or hydrolyzed asdescribed above to yield Compound 2. Moreover, the use of targetingagents may limit the dosage required to be administered in order toachieve an effective concentration of the drug at the site of treatment.The use of targeting agents may also allow for more infrequent dosage oreven alternative methods of administration in order to achieve aneffective concentration of the drug at the site of treatment.

The targeting agent are preferentially attached to the compounds of thepresent invention via a covalent bond which may be formed by methodsincluding, but not limited to, a nucleophilic or electrophilic group ofthe targeting agent that is covalently reacted with an electrophilic ornucleophilic group (respectively) on the linker.

In one embodiment of the present invention, Compounds 2-3 of the presentinvention are those compounds wherein, R₆-T is selected from the groupconsisting of the following:

Targeting agents which may be reacted with the prodrugs of the presentinvention include, but are not limited to, carbohydrates, vitamins,peptides, proteins, nucleosides, nucleotides, nucleic acids, liposomes,lipids, bone-seeking agents and cartilage-seeking agents. The targetingagent may also be a molecule which is bound by a receptor in a desiredtissue and optionally transported into a cell by a receptor-mediatedprocess. Representative examples of such targeting agents include, butare not limited to, diazepines that bind to peripheral benzodiazepinereceptors (PBRs) present in glial cells in the brain. Representativeexamples of such diazepines are discussed in G. Trapani, et al.Bioconjugate Chem. 2003, vol 14, pp 830-839 “Peripheral BenzodiazepineReceptor Ligand-Melphalan Conjugates for Potential Selective DrugDelivery to Brain Tumors,” the contents of which are incorporated byreference.

Representative vitamins that may be used as targeting agents include,but are not limited to, folate, vitamin B₁₂ or vitamin C. The term“folate” encompasses folic acid derivatives with capacity to bind withfolate-receptors. Representative examples of folates that may be used astargeting agents include, but are not limited to, folic acid, folinicacid, pteropolyglutamic acid, and folate receptor-binding pteridinessuch as tetrahydropterins, dihydrofolates, tetrahydrofolates and theirdeaza and dideaza analogs. Other suitable folates are folate analogsincluding, but not limited to, aminopterin, amethopterin (methotrexate),N₁₀-methylfolate, 2-deamino-hydroxyfolate, deaza analogs such as1-deazamethopterin or 3-deazamethopterin, and3′5′-dichloro4-amino-4-deoxy-N₁₀-methylpteroyl-glutamic acid(dichloromethotrexate). Methods of conjugating molecules to folates thatare suitable for covalent attachment to compounds of the presentinvention are disclosed in U.S. Pat. Nos. 6,576,239, 5,820,847,5,688,488, 5,108,921, 5,635,382, and 5,416,016 the contents of which areincorporated herein by reference. Methods of conjugating molecules tovitamin C that are suitable for covalent attachment for compound of thepresent invention are disclosed in S. Manfrdini J. Med. Chem. Vol 45, pp559-562, 2002 the contents of which are incorporated herein byreference.

Representative peptides and peptidomimetics that may be used astargeting agents include, but are not limited to, an RGD-containingpeptide selected from the group consisting of RGDs, c(RGDfK),vitronectin, fibronectin, somatostatin-receptor agonists andsomatostatin-receptor antagonists. Molecules that bind to the avb3integrin receptor and act as antagonists may be used at targeting agentsas described in U.S. Pat. Nos. 6,552,079, 6,426,353B, WO 2002/40505A2,and U.S. Patent Publications 2002/0055499, 2002/0061885, 2002/0065291,2002/0072500, U.S. 2002/0072518; W. Arap et al. Science vol 279, number16, 1998, pp 377-380; R J Kok et al. Biojonjugate Chem. 2002, vol 13, pp128-135; D A Sipkins et al. Nature Medicine vol 4, number 5, 1998 pp623-626; P M Winter et al. Cancer Research 2003, vol 63, pp 5838-5843;and J D Hood et al. Science vol 296, pp 2404-2407; the contents of whichare incorporated herein by reference. Representative proteins that maybe used as targeting agents include, but are not limited to, antibodiesor fragments thereof, such as a tumor-specific monoclonal antibody orfragment thereof. Representative bone-seeking agents that may be used astargeting agents include, but are not limited to, phosphonate,phosphonic acid, aminomethylphosphonic acid, phosphate, polyphosphate,and hydroxyapatite-binding polypeptides. Other peptides includechlorotoxin (SU6,429,187B1) and tissue factor (G. M. Lanza, et al.“Targeted Antiproliferative Drug Delivery to Vascular Smooth MuscleCells with a Magnetic Resonance Imaging Nanoparticle Contrast Agent”;Circulation, 2002 volume 106 pp 2842-2847).

Other suitable targeting agents include antibodies. The antibodies maybe of classes IgG, IgM, IgA, IgD or IgE, or fragments or derivativesthereof, including Fab, F(ab′)₂, Fd, and single chain antibodies,diabodies, bispecific antibodies, bifunctional antibodies andderivatives thereof. The antibody may be a monoclonal antibody,polyclonal antibody, affinity purified antibody, or mixtures thereofwhich exhibits sufficient binding specificity to a desired epitope or asequence derived therefrom. The antibodies may also be a chimericantibody. The antibodies may be directed against a variety of antigenicdeterminants including those associated with tumors, histocompatibilityand other cell surface antigens, bacteria, fungi, viruses, enzymes,toxins, drugs and other biologically active molecules. Antigensassociated with tumors for which antibodies may be specifically reactiveinclude, but are not limited to, such antigens as are and include, butare not limited to, carcinoembryonic antigen (CEA), mucins such asTAG-72, human milk fat globule antigens, prostate serum antigent (PSA),prostate specific membrane antigen (PSMA), PS (phosphatidyl serine), andreceptors including, but not limited to, the IL-2, EGF, VEGF andtransferrin receptors. Other representative antigens associated withtumors include, but are not limited to, those tumor associated antigensdescribed in Zalcberg and McKenzie, J. Clin. Oncology, Vol. 3; pp.876-82 (1985), WO 01/68709A1, and U.S. Patent PublicationUS2004/0009122A1, the contents of which are incorporated herein byreference.

Other suitable targeting agents include glucose, galactose, mannose,mannose 6-phosphate, hormones (e.g., insulin, growth hormone, and thelike), growth factors or cytokines (e.g., TGFβ, EGF, insulin-like growthfactor, and the like), YEE(GalNAcAH).sub.3 or derivatives, cobalamin,α-2 macroglobulins, asialoglycoprotein, albumin, texaphyrin,metallotexaphyrin, antibodies, antibody fragments (e.g., Fab),single-chain antibody variable region (scFv), transferrin, any vitaminand any coenzyme

The targeting agent may also be an agent that delivers the prodrug tobones. Bone targeting agents include, but are not limited to, EDTMPDOTMP, and ABEDTMP, which are disclosed in U.S. Pat. Nos. 4,937,333,4,882,142, 5,064,633 and WO-94/00143, the contents of which areincorporated herein by reference. DOTMP and EDTMP may be attached to thelinker moiety by any method including, but not limited to, the couplingchemistry shown in FIG. 3 and the alkylation chemistry shown in FIG. 4where the R group can have an appropriate electrophilic or nucleophilicgroup that reacts with the nucleophilic or electrophilic (respectively)group of the linker moiety. Further details of the coupling chemistryare provided in Tetrahedron 1999, 55, pp 12997-13010, the contents ofwhich are incorporated by reference. Further details of the alkylationchemistry are provided in Proc. SPIE-Int. Soc. Opt. Eng. 1999, 3600(Biomedical Imagn. Reporters Dyes & Instrumental, pp 99-106; U.S. Pat.No. 5,177,054; J Med. Chem. 1994, 37, 498-511; Tetrahedron Letters,1989, 30 #51 pp 7141-7144; and U.S. Pat. No. 5,955,453, the contents ofwhich are incorporated by reference.

The targeting agent may be used to deliver the prodrug to bones as aslow release reservoir site for the compounds of the present invention.The targeting agent may be a bone seeking (osteotropic) moiety attachedto the compounds of the present invention via an acid cleavable linkerattached to the quaternary amine. Examples of an acid cleavable linkerinclude, but are not limited to, an ortho acid-amide linkage. Underacidic conditions the protein-ACL-3 amide linkage is readily cleavedfreeing the native amino group of the amide functionality as describedin WO-94/00143 the contents of which are incorporated by reference.During osteoclastic bone resorption, which involves an acidic mediatedmechanism, the attachment tethering the prodrug to bone may be cleavedreleasing the compounds of the present invention.

The targeting agent used to deliver the prodrugs to bones may be amolecule that binds with notch receptors. Notch signaling plays a keyrole in the development and differentiation of various hematopoieticlineages. As discussed in Jundt et al., Blood, 102(11): 928a (2003),ligand-induced notch signaling is a novel growth factor for multiplemyeloma cells and suggests that these interactions contribute tolymphomagenesis of multiple myeloma in vivo.

The bone targeting agent may have a high affinity for calcium ions inhydroxyapatite, the major constituent of bone. The compound of theinvention can be targeted to calcium deposits in regions of the bodyother than bone, such as calcium deposits in the arteries, heart,kidney, or gall bladder. However, the bone targeting agent ideallyselectively binds to bone tissue. A bone targeting agent of theinvention is attracted to the bone tissue of the subject, preferablybinds to the bone with a higher affinity than non-bone tissues, andremains bound for a certain length of time thereby delivering thecomposition to a bone environment. In other words, the bone targetingagent preferably binds to bone tissue with at least 2-fold greateraffinity (e.g., at least 3-fold, at least 5-fold, at least 10-fold, orat least 25-fold greater affinity) than the bone targeting agent bindsto non-bone tissue. The bone targeting agent reversibly binds to bonetissue, meaning that the bone targeting agent is eventually releasedfrom bone and expelled from the body.

The bone targeting agent may remain bound to bone tissue for asufficient period of time to allow the quaternary prodrug to behydrolyzed, thereby delivering the active drug to the target cells(e.g., bone marrow cells). The bone targeting agent can remain bound tobone for about 1 day (e.g., about 2 days, about 3 days, or about 7 days)to about 1 year (e.g., about 330 days, about 365 days, or about 400days), after which the bone targeting agent is expelled from the body.The bone targeting agent can remain bound to bone for about 7 days(e.g., about 7 days, about 14 days, or about 21 days) to about 6 months(e.g., about 90 days, about 120 days, or about 150 days). For example, abone targeted prodrug can remain bound to the bone for 30 days, duringwhich time the drug is released. After about 45 days the bone targetingagent would be released from the bone and eventually excreted. Thus, abone targeting agent for use in the invention can be selected based onbinding kinetics to bone tissue. Candidate bone targeting agents can bescreened in vitro by determining affinity to bone tissue (e.g.,hydroxyapatite) in, for example, a multi-well format. Candidate bonetargeting agents also can be screened in vivo by assessing the rate andtiming of excretion of candidate bone targeting agents from the body. Inthis respect, the bone targeting agent preferably is expelled from thebody via the kidneys.

The bone targeting agent desirably is selected from the group consistingof a phosphate, a phosphonate, a bisphosphonate, ahydroxybisphosphonate, an aminomethylenephosphonic acid, and an acidicpeptide. The bone targeting agent of the invention can carry one, morethan one, or a mixture of these groups. For example, the bone targetingagent can be a phosphonate, meaning that the bone targeting agent maycomprise one phosphonate, two phosphonates, or three or morephosphonates. One suitable bone targeting agent for use in the inventionis EDTMP (ethylene diamine-N,N,N′,N′-tetrakis(methylenephosphonic acid),the chemical structure of which is set forth in FIG. 1, currently FDAapproved (Quadramet™) as the radioactive ¹⁵³Sm complex for delivering aselective radiation dose to bone metastases for pain palliation. EDTMPis a phosphonate that contains four phosphonic acid groups, and istherefore a tetraphosphonate. Compounds such as ¹⁵³Sm-EDTMP areselectively localized in bone where tumors are present versus normalbone in a ratio of more than 10:1, probably because metabolic turnoverof calcium is very high in the metastatic region. The ¹⁵³Sm-EDTMPreportedly is rapidly taken up by the skeleton in osteoblastic bonemetastases and cleared from the plasma. That portion of the compoundthat does not accumulate in the skeleton reportedly is rapidly excreted,and excretion is almost complete within 6 hours after administration(Jimonet et al., Heterocycles, 36, 2745 (1993)). The pain palliation isthought to be due to the radiation originating from the isotope bound tothe osteoblastic bone metastases having some effect on the nearbymetastatic tumor cells. Another clinically useful bone-targeting systemis DOTMP (the chemical structure of which is set forth in FIG. 1, now inPhase III clinical trials (termed STR, skeletal targeted radiation) asthe radioactive ¹⁶⁶Ho complex designed to deliver large doses ofradiation selectively to the bone marrow for the treatment of multiplemyeloma. It should be noted that the radioactive ¹⁶⁶HO-DOTMP complexlocalizes in the skeletal system and irradiates the nearby bone marrowwhich houses the malignant myeloma cells. Like the ¹⁵³Sm-EDTMP system,the phosphonate that does not localize in the bone is cleared throughthe urine and out the body. In general, the skeletal uptake is about 20to about 50% of the injected dose, and the localization in areas of theskeleton with tumor infiltration is illustrated in FIG. 7 of Bayouth etal., J. Nucl. Med., 36; 730 (1995).

Preferably, the bone targeting agent is a polyphosphonic acid.Polyphosphonic acid has been demonstrated to successfully targetbiologically-active molecules to bone tissue. For example, conjugation(via isothiocyanato chemistry) of polyaminophosphonic acids, such asABDTMP (the chemical structure of which is set forth in FIG. 1, togrowth factors (to stimulate bone formation) successfully resulted inthe targeting of the growth factors to the bones of rats (see, forexample, International Patent Application WO 94/00145). Similarly, bonetargeting agents have been coupled to proteins. For examplebisphosphonates that were conjugated to human serum albumin successfullydelivered the protein to bone in vitro (Biotechnol. Prog., 16, 258(2000)) and in vivo (Biotechnol. Prog., 16, 1116 (2000)). The utility ofbone-seeking agents extends beyond delivery of proteins to bone andincludes, for instance, small therapeutic molecules. A conjugatecomprising a bone-seeking bisphosphonate and an alkylating agent, suchas BAD (the chemical structure of which is set forth in FIG. 1, has beengenerated (see, for example, Wingen et al., J. Cancer Res. Clin. Oncol.,111, 209 (1986)). In this molecule, the alkylating agent is not specificin its interaction with its target (DNA), and, thus, there is norequirement for cleavage between the bisphosphonate (i.e., bone-seekingagent) and the alkylating moiety. The bisphosphonate-alkylating agentdemonstrated efficacy in a rat osteosarcoma model using BAD. Anotherseries of studies have been performed using the antifolateantineoplastic agent methotrexate that has been covalently attached tobisphosphonates, designated MTX-BP and shown in FIG. 1 (see, forexample, Sturtz et al., Eur. J. Med. Chem., 27, 825 (1992); Sturtz etal., Eur. J. Med. Chem., 28, 899 (1993); and Hosain et al., J. Nucl.Med., 37, 105 (1996)). Using Tc-99m labeled MTX-BP, it was determinedthat around 15% of the injected dose was localized in the skeleton after4 hours with about 61% of the dose being excreted (Hosain, supra).MTX-BP further demonstrated five times greater anticancer activitycompared with methotrexate alone in animal models of transplantedosteosarcoma (Sturtz 1992, supra). Similar work has been described usingthe conjugate CF—BP, a carboxyfluorescein group with an appendedbisphosphonate whose chemical structure is set forth in FIG. 1 (Fujisakiet al., Journal of Drug Targeting, 4, 117 (1994)). In this molecule, theCF group is a fluorescent marker to quantitate pharmacokinetics andbiodistribution, and is connected to the bone targeting agent through anester bond which is susceptible to hydrolysis in vivo. Studies in ratsinjected intravenously indicated that CF—BP localized in the bone andserved as a slow release mechanism for CF generated via generalhydrolysis of the ester linkage (Fujisaki, supra).

In another embodiment, the bone-seeking agent can be a peptide, such as(Asp)₆ and (Glu)₆. The acid-rich peptide sequence of the glycoproteinosteonectin, which is found in abundance in bone and dentin, has astrong affinity to hydroxyapatite (Fujisawa et al., Biochimica etBiophysica Acta, 53, 1292 (1996)). Thus, peptide ligands comprisingacidic amino acids are ideal candidates for bone targeting agents.Indeed, (Glu)₁₀, when attached to biotin, successfully recruited labeledstrepavidin to hydroxyapatite (described further in Chu and Orgel,Bioconjugate Chem., 8, 103 (1997), and International Patent ApplicationWO 98/35703). In addition, the biological half-life of the fluoresceinisothiocyanate conjugated to (Asp)₆ was 14 days in the femur (Kasugai etal., Journal of Bone and Mineral Research, 15(5), 936 (2000)), which isan acceptable half-life for the bone targeting agent of the invention.Likewise, delivery of estradiol-(Asp)₆ conjugates to bone has beendemonstrated in ovariectomized animals with concomitant inhibition ofosteoporectic-type bone loss (Kasugai et al., Journal of Bone andMineral Research (Suppl 1), 14, S534 (1999)). It is believed that the(Asp)₆ tether to bone is metabolized during the bone resorption processmediated by osteoclasts. Therefore, the acidic peptide ligand providesnot only a means of recruiting compounds to bone, but also provides amechanism of slowly releasing compounds to bone cells and surroundingtissue.

Other examples of bone targeting agents include, but are not limited toamino- and hydroxy-alkyl phosphonic and diphosphonic acids;hydroxybisphosphonic acids including alendronate, pamidronate,4-aminobutylphosphonic acid, 1-hydroxyethane-1,1-diphosphonic acid, andaminomethylenebisphosphonic acid; phosphates such as phytic acid; andaminomethylenephosphonic acids such asN,N-bis(methylphosphono)-4-amino-benzoic acid andnitrilotri(methylphosphonic acid). Nonlimiting examples of some bonetargeting agents are shown in FIG. 2.

Preferably, the bone targeting agent is an aminomethylenephosphonicacid. By “aminomethylenephosphonic acid” is meant a compound thatcontains an —NCH₂PO₃H moiety, where the amino group has one, two, orthree methylenephosphonic acid groups attached, and may be furthersubstituted with other chemical moieties. An aminomethylenephosphonicacid may include one or more phosphonic acid groups and one or moreamino groups. Examples of these aminomethylenephosphonic acids includebut are not limited to the compounds F through N set forth in FIG. 2.

It is envisioned that these bone targeting agents and other bonetargeting agents can be attached through one of the heteroatoms or bychemical modification that installs an additional attachment point. Forexample, EDTMP can be connected to a linker by one of the phosphorousoxygens to create a phosphonate linkage, as illustrated in FIG. 3 (seefor example Vieira de Almedia et al., Tetrahedron, 55, 12997-13010(1999).) The phosphorous oxygen can also be alkylated as shown in FIG.4, where the R group can have, for example, a pendant amino group, toprovide a secondary attachment point for ligation to, for example, anactivated PEG. Other types of alkylation that could be utilized in theinvention include but are not limited to examples similar to thatinvolving DOTMP, as has been further described in Chavez et al.,Biomedical Imaging Reporters, Dyes, & Instumentation, Contag &Sevick-Muracia, Eds., Proc. SPIE, Vol. 3600, 99-106 (July, 1999), or asshown for other phosphonic acids further described in, for example, U.S.Pat. No. 5,177,064, U.S. Pat. No. 5,955,453, de Lombaert et al., J Med.Chem., 37, 498-511 (1994), and Iyer et al., Tetrahedron Letters, 30(51),7141-7144 (1989). Alternatively, for chemical modification, EDTMP canbe, for example, modified to generate ABDTMP by installation of ananiline group (as further described in, for example, FIG. 1 ofInternational Patent Application WO 94/00145). The aniline amine is thenavailable to form, for example, an amide bond. DOMTP could be similarlymodified, as outlined in FIG. 5.

The terms “phosphonate, phosphate, and aminomethylenephosphonate” aremeant to encompass the phosphonic acids, the phosphoric acids, andaminomethylenephosphonic acids, respectively, as well as any salts,hydrolyzable esters, and prodrugs of the phosphorous-based acidsthereof. At the biological pH of 7.4 in the blood, or the more acidic pHaround the bone, a certain portion of the phosphate or phosphonate ofthe bone targeting agent may be deprotonated and replaced with acounterion. Furthermore, the exchange of proton for calcium is aninherent event for the binding of the bone targeting agent to thehydroxyapatite in the invention. However, preparation and administrationof the composition containing the bone targeting agent may or may notrequire complete protonation of the phosphorous acids therein.Therefore, the phosphonic acid, phosphoric acid, andaminomethylenephosphonic acid are drawn and utilized interchangeablywith phosphate, phosphonate, and aminomethylenephosphonate. While notparticularly preferred, biologically hydrolyzable esters of thephosphorus-based acids may also be utilized in the in vivo use of thebone targeting prodrugs. Similarly, prodrugs of the phosphorous-basedacids may also be utilized in vivo to mask the acidity of thecomposition during, for example, formulation and administration.

The targeting agent may also be an agent that targets based uponproperties of the particular tissue. Representative examples of suchtargeting agents include, but are not limited to, polymers that areselectively localized in tumor tissues due to the EPR effect (enhancedpermeability and retention) as described in H. Maeda et al “Tumorvascular permeability and the EPR effect in macromolecular therapeutics:A Review”; Journal of Controlled Release, 2000 vol 63, pp 271-284, thecontents of which are incorporated by reference. Other representativepolymers are N-(2-hydroxypropyl)methacrylamide (HPMA) and(poly)L-glutamic acids.

The targeting agent may also comprise an RGD moiety. As discussed inCurnis et al., Cancer Research, 64(2): 565-571 (2004), RGD moietiestarget RGD fusion proteins to vasculature by interacting with interactswith cell adhesion receptors, including α_(v)β₃ integrin.

3. SYNTHESIS

a. Main Ring System

The compounds of the present invention may be synthesized using LY294002(Compound 1) as a starting product. LY294002 (Compound 1) may beobtained commercially or synthesized as described in Example 1 or asdescribed in U.S. Pat. No. 5,703,075, the contents of which areincorporated herein by reference. One of ordinary skill in the art mayalso synthesize the compounds of the present invention using Compound 2as a starting product.

b. Preparation of Derivatives of Main Ring System

The main ring system of Compounds 2 and 3 may be derivatives of the mainring system of LY294002 (Compound 1). Derivatives of the main ringsystem of Compound 3 may be prepared as disclosed in U.S. Pat. No.5,703,075, the contents of which are incorporated herein by reference,for the preparation of main ring derivatives of LY294002 (Compound 1).Derivatives of the main ring system of Compound 3 may also be preparedby using commercially available compounds including, but not limited to,substituted 2-hydroxy-acetophenones.

c. Preparation of Derivatives of Morpholine Ring

The amine derivatives of Compound 3 may be prepared by the displacementof the thioalkyl group in Example 1 under conditions ranging from roomtemperature to forcing conditions (excess nucleophile and heating to110° C.). Any primary or secondary nitrogen-containing nucleophile mayreact to give alternative amine substitutions to the morpholine ringstructure (including different morpholine analogs). The synthesis ofrepresentative examples of such amine derivatives of Compound 3 aredescribed in the Examples herein.

d. Preparation of Esters

As described above, esters may be used to form the quaternized compoundsof the present invention. The quaternized compounds of the presentinvention are preferably formed using halo esters. In one preferredembodiment, the quaternized compounds of the present invention areformed using chloromethyl esters. Numerous chlorlomethyl esters usefulin the preparation of the compounds of the present invention areavailable from commercial sources. In addition, chloromethyl esters maybe synthesized as described in WO 02/42265, WO 94/23724, and U.S. Pat.Nos. 4,444,686, 4,264,765, and 4,342,768, the contents of which areincorporated herein.

e. Quaternization

The prodrugs of the present invention may be prepared by quaternizingthe tertiary amine of Compound 1 or Compound 2 with a halomethyl ester,for example, as described in Example 4 and Example 6. Quaternized aminecompounds are generally not reversible under mild conditions. However,the quaternary compounds of the present invention are readilyhydrolyzable as discussed above. Halomethyl esters that may be used toquaternize the tertiary amine of Compound 1 or Compound 2 arecommercially available or may be prepared as described in the Examplesbelow.

f. Linkers

The prodrugs of the present invention may also be prepared byquaternizing the tertiary amine of Compound 1 or Compound 2 with alinker comprising at least two functional groups. The linker may be anynatural or synthetic linker that is capable of quaternizing the tertiaryamine and is also capable of being covalently attached to a targetingmolecule or may already be attached to a targeting molecule.

Linkers are preferably comprised of an atom such as oxygen or sulfur, aunit such as —NH—, —CH₂—, —C(O)—, —C(O)NH—, or a chain of atoms. Themolecular mass of a linker is typically in the range of about 14 to 200,preferably in the range of 14 to 96 with a length of up to about sixatoms. Representative examples of linkers include but are not limited toa saturated or unsaturated aliphatic group which is optionallysubstituted, and wherein one or two saturated carbons of the chain areoptionally replaced by —C(O)—, —C(O)C(O)—, —CONH—, —CONHNH—, —C(O)O—,—OC(O)—, —NHCO₂—, —O—, —NHCONH—, —OC(O)NH—, —NHNH—, —NHCO—, —S—, —SO—,—SO₂—, —NH—, —SO₂NH—, or —NHSO₂—.

The first functional group of the linker is used to quaternize thetertiary amine as discussed above. A preferred first functional group isa halomethyl ester including, but not limited to, chloromethylester andiodomethyl ester. The second functional group of the linker may be usedto covalently attach a targeting agent.

The second functional group may be an electrophilic group or anucleophilic group. Preferred second functional groups for covalentlyattaching targeting groups are isothiocyanate, haloacetamide maleimide,imidoester, thiophthalimide, N-hydroxysuccinimyl ester, pyridyldisulfide, phenyl azide, carboxyl (and acid chlorides thereof), amino,acyl hydrozide, semicarbazide, thiosemicarbazide, diazonium, hydrazine,azide, aminoalkylurea, aminoalkylthiourea, halotriazine, and meta(dihydroxyboryl)phenylthiourea. Other suitable reactive moieties whichmay be suitable for covalently attaching the prodrugs of the presentinvention to targeting agents include disulfides, nitrenes,sulfonamides, carbodiimides, sulfonyl chlorides, benzimidates, —COCH₃and —SO₃H.

The appropriate second functional group will depend on the functionalgroup of the targeting agent with which the covalent bond will be formedand by its susceptibility to loss of biological activity as aconsequence of forming a given type of linkage. If the targeting agentis a protein, the second functional group may be reactive with sidechain groups of amino acids making up the polypeptide backbone. Suchside chain groups include the carboxyl groups of aspartic acid andglutamic acid residues, the amino groups of lysine residues, thearomatic groups of tyrosine and histidine, and the sulfhydryl groups ofcysteine residues.

Carboxyl side groups presented by a targeting agent such as apolypeptide backbone may be reacted with amine second functional groupsby means of a soluble carbodiimide reaction. Amino side groups presentedby a targeting agent may be reacted with isothiocyanate, isocyanate orhalotriazine second functional groups to effect linkage to the prodrugsof the present invention. Alternatively, amino side groups on thetargeting agent may be linked to the prodrugs compounds of thisinvention bearing amine reactive groups by means of bifunctional agentssuch as dialdehydes and imidoesters. Aromatic groups presented by atargeting agent may be coupled to the prodrugs of this invention viadiazonium derivatives. Sulfhydryl groups on targeting agent moleculesmay be reacted with maleimides or with haloalkyl targeting agentreactive groups such as iodoacetamide. Free sulhydryl groups suitablefor such reactions may be generated from the disulfide bonds of proteinimmunoglobulin or may be introduced by chemical derivatization. Linkageto free sulfhydryl groups generated in the intra-heavy chain region ofimmunoglobulins does not interfere with the antigen binding site of theimmunoglobulin but may render the antibody incapable of activatingcomplement.

When the targeting agent is a glycosylated protein, an alternative toforming a linkage to the compounds of the present invention via thepolypeptide backbone is to form a covalent linkage with the carbohydrateside chains of the glycoprotein according to the methods such as thoseof McKearn, et al., EPO 88,695. Thus, the carbohydrate side chains ofantibodies may be selectively oxidized to generate aldehydes which maythen be reacted either with amine reactive groups to form a Schiff baseor with hydrazine, semicarbazide or thiosemicarbazide reactive groups,to give the corresponding hydrazone, semicarbazone or thiosemicarbazonelinkages. These same methods may also be employed to link the prodrugsof this invention to non-proteinaceous targeting agents such ascarbohydrates and polysaccharides.

An alternative targeting agent reactive moiety useful for linkage tocarbohydrates and polysaccharides without the necessity for prioroxidation is the dihydroxyboryl groups, such as is present in meta(dihydroxyboryl)phenylthiourea derivatives. This group is reactive withtargeting agents containing a 1,2-cis-diol, forming a 5-membered cyclicborate ester, and thus is of use with those carbohydrates,polysaccharides and glycoproteins which contain this group. Thedihydroxyboryl derivatives may also be used to link the prodrugs of thisinvention to ribonucleosides, ribonucleotides and ribonucleic acids,since ribose contains a 1,2-cis-diol group at the 2′,3′ position, asdisclosed by Rosenberg, et al., Biochemistry, 11, 3623-28 (1972).Deoxyribonucleotides and DNA targeting agents may not be linked to thepresent prodrugs in this fashion as the 3′ hydroxyl group is absent. Thelatter targeting agents may, however, be conjugated to isothiocyanatederivatives of prodrugs by first forming an allylamine derivative of thedeoxyribonucleotide as disclosed by Engelhardt, et al., EPO 97,373.

When the targeting agent to be linked with the prodrugs of thisinvention is an intact cell, either polypeptide reactive or carbohydratereactive moieties may be employed. Hwang and Wase, Biochim. Biophys.Acta, 512, 54-71 (1978), disclose the use of the diazonium derivative ofthe bifunctional EDTA chelator of Sundberg, et al., J. Med. Chem., 17,1304 (1974), to label erythrocytes and platelets with indium-111. Thedihydroxyboryl group is reactive with a variety of bacteria, viruses andmicroorganisms, see Zittle, Advan. Enzym., 12 493 (1951) and Burnett, etal., Biochem. Biophys. Res. Comm., 96, 157-62 (1980).

Preferred linkers that may be used to covalently quaternize the tertiaryamine of Compound 1 or Compound 2 and are of the formula:

wherein,

-   -   X represents a halo group;    -   Y represents —CH₂—, —CH(CH₃)—, —CH(Ph)-, —C(CH3)(COOH)— or        CH(CH(CH3)2)—    -   Z₁ and Z₂ independently are S or O; and    -   n=0 to 4.

In one embodiment, Compound 4 of the present invention are thosecompounds wherein,

-   -   X represents Cl or I;    -   Y represents —CH₂—, —CH(CH₃)—, —CH(Ph)-, —C(CH3)(COOH)— or        CH(CH(CH3)2)—;    -   Z₁ and Z₂ independently are O; and    -   n=0.

In another embodiment, Compound 4 of the present invention are thosecompounds wherein,

-   -   X represents Cl or I;    -   Y represents —CH₂—, —CH(CH₃)—, —CH(Ph)-, —C(CH3)(COOH)— or        CH(CH(CH3)2)—;    -   Z₁ and Z₂ independently are O; and    -   n=1.

Compound 4 provides linkers with both an alkyl and aryl carboxylicbackbone which provides flexibility in the cleavage rate of the finalquaternary nitrogen. The linkers of Compound 4 may be prepared usingcommercially available starting products as described in Example 5.

g. Purification

The compounds of the present invention may be isolated using standardpurification methods. The hydrolyzable bond of the compounds of thepresent may be prone to hydrolysis during the purification of thecompounds.

The present invention is also directed to methods of purifying thecompounds of the present invention comprising adding the compounds to asolution comprising at least 0.1% acid (v/v) to solubilize the compound.The compound is then purified by performing chromatography, preferablyHPLC.

h. Testing

The prodrugs of the present invention may be tested to determine therate of hydrolysis of the hydrolyzable bond and the products ofhydrolysis by performing HPLC analysis of the prodrug exposed tocleavage conditions as a function of time. The biological activity ofthe compounds of the present invention may be measured by methodsincluding, but not limited to, blocking phagocytosis in macrophage cellline J774 cells as described in Example 17. The biological activity ofthe compounds of the present invention may also be measured by PI-3kinase enzyme assays as described by U.S. Pat. No. 5,480,906; K.Fuchikami et al J. Biomol Screen, 2002 October pp 441-450; V I Silveriaet al J. Biomol. Screen, 2002, Dec. 7(6), 507-514; BE DreesCombinatorial Chemistry and Highthroughput Screening 2003, vol 6,321-330, the contents of which are incorporated by reference.

i. Salts

The compounds of the present invention are useful in variouspharmaceutically acceptable salt forms. The term “pharmaceuticallyacceptable salt” refers to those salt forms which would be apparent tothe pharmaceutical chemist, i.e., those which are substantiallynon-toxic and which provide the desired pharmacokinetic properties,palatability, absorption, distribution, metabolism or excretion. Otherfactors, more practical in nature, which are also important in theselection, are cost of the raw materials, ease of crystallization,yield, stability, hygroscopicity and flowability of the resulting bulkdrug. Conveniently, pharmaceutical compositions may be prepared from theactive ingredients or their pharmaceutically acceptable salts incombination with pharmaceutically acceptable carriers.

Pharmaceutically acceptable salts of the compounds of the presentinvention which are suitable for use in the methods and compositions ofthe present invention include, but are not limited to, salts formed witha variety of organic and inorganic acids such as hydrogen chloride,hydroxymethane sulfonic acid, hydrogen bromide, methanesulfonic acid,sulfuric acid, acetic acid, trifluoroacetic acid, maleic acid,benzenesulfonic acid, toluenesulfonic acid, sulfamic acid, glycolicacid, stearic acid, lactic acid, malic acid, pamoic acid, sulfanilicacid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid,methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethonicacid, and include various other pharmaceutically acceptable salts, suchas, e.g., nitrates, phosphates, borates, tartrates, citrates,succinates, benzoates, ascorbates, salicylates, and the like. Cationssuch as quaternary ammonium ions are contemplated as pharmaceuticallyacceptable counterions for anionic moieties.

Preferred salts of the compounds of the present invention includehydrochloride salts, methanesulfonic acid salts and trifluoroacetic acidsalts with methanesulfonic acid salts being more preferred. In addition,pharmaceutically acceptable salts of the compounds of the presentinvention may be formed with alkali metals such as sodium, potassium andlithium; alkaline earth metals such as calcium and magnesium; organicbases such as dicyclohexylamine, tributylamine, and pyridine; and aminoacids such as arginine, lysine and the like.

The pharmaceutically acceptable salts of the present invention can besynthesized by conventional chemical methods. Generally, the salts areprepared by reacting the free base or acid with stoichiometric amountsor with an excess of the desired salt-forming inorganic or organic acidor base, in a suitable solvent or solvent combination.

In general, the counterions of the salts of the compounds of the presentinvention are determined by the reactants used to synthesized thecompounds. There may be a mixture of counterions of the salts, dependingon the reactants. For example, where NaI is added to facilitate thereaction the counterion may be a mixture of Cl and I counter anions.Furthermore preparatory HPLC may cause the original counterion to beexchanged by acetate anions when acetic acid is present in the eluent.The counterions of the salts may be exchanged to a different counterion.The counterions are preferably exchanged for a pharmaceuticallyacceptable counterion to form the salts described above. Procedures forexchanging counterions are described in WO 2002/042265, WO 2002/042276and S. D. Clas, “Quaternized Colestipol, an improved bile saltadsorbent: In Vitro studies.” Journal of Pharmaceutical Sciences, 80(2):128-131 (1991), the contents of which are incorporated herein byreference. For clarity reasons the counterions are not explicitly shownin the chemical structures herein and the characterization of thecompounds is based on the identified quarternary cation.

4. COMPOSITION

The present invention also encompasses a composition comprising one ormore compounds of the present invention. The compositions of the presentinvention may further comprise one or more pharmaceutically acceptableadditional ingredient(s) such as alum, stabilizers, antimicrobialagents, buffers, coloring agents, flavoring agents, adjuvants, and thelike.

a. Formulation

Compositions of the present invention may be in the form of tablets orlozenges formulated in a conventional manner. For example, tablets andcapsules for oral administration may contain conventional excipientsincluding, but not limited to, binding agents, fillers, lubricants,disintegrants and wetting agents. Binding agents include, but are notlimited to, syrup, accacia, gelatin, sorbitol, tragacanth, mucilage ofstarch and polyvinylpyrrolidone. Fillers include, but are not limitedto, lactose, sugar, microcrystalline cellulose, maizestarch, calciumphosphate, and sorbitol. Lubricants include, but are not limited to,magnesium stearate, stearic acid, talc, polyethylene glycol, and silica.Disintegrants include, but are not limited to, potato starch and sodiumstarch glycollate. Wetting agents include, but are not limited to,sodium lauryl sulfate). Tablets may be coated according to methods wellknown in the art.

Compositions of the present invention may also be liquid formulationsincluding, but not limited to, aqueous or oily suspensions, solutions,emulsions, syrups, and elixirs. The compositions may also be formulatedas a dry product for constitution with water or other suitable vehiclebefore use. Such liquid preparations may contain additives including,but not limited to, suspending agents, emulsifying agents, nonaqueousvehicles and preservatives. Suspending agent include, but are notlimited to, sorbitol syrup, methyl cellulose, glucose/sugar syrup,gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminumstearate gel, and hydrogenated edible fats. Emulsifying agents include,but are not limited to, lecithin, sorbitan monooleate, and acacia.Nonaqueous vehicles include, but are not limited to, edible oils, almondoil, fractionated coconut oil, oily esters, propylene glycol, and ethylalcohol. Preservatives include, but are not limited to, methyl or propylp-hydroxybenzoate and sorbic acid.

Compositions of the present invention may also be formulated assuppositories, which may contain suppository bases including, but notlimited to, cocoa butter or glycerides. Compositions of the presentinvention may also be formulated for inhalation, which may be in a formincluding, but not limited to, a solution, suspension, or emulsion thatmay be administered as a dry powder or in the form of an aerosol using apropellant, such as dichlorodifluoromethane or trichlorofluoromethane.Compositions of the present invention may also be formulated transdermalformulations comprising aqueous or nonaqueous vehicles including, butnot limited to, creams, ointments, lotions, pastes, medicated plaster,patch, or membrane.

Compositions of the present invention may also be formulated forparenteral administration including, but not limited to, by injection orcontinuous infusion. Formulations for injection may be in the form ofsuspensions, solutions, or emulsions in oily or aqueous vehicles, andmay contain formulation agents including, but not limited to,suspending, stabilizing, and dispersing agents. The composition may alsobe provided in a powder form for reconstitution with a suitable vehicleincluding, but not limited to, sterile, pyrogen-free water.

Compositions of the present invention may also be formulated as a depotpreparation, which may be administered by implantation or byintramuscular injection. The compositions may be formulated withsuitable polymeric or hydrophobic materials (as an emulsion in anacceptable oil, for example), ion exchange resins, or as sparinglysoluble derivatives (as a sparingly soluble salt, for example).

Compositions of the present invention may also be formulated as aliposome preparation. The liposome preparation can comprise liposomeswhich penetrate the cells of interest or the stratum corneum, and fusewith the cell membrane, resulting in delivery of the contents of theliposome into the cell. For example, liposomes such as those describedin U.S. Pat. No. 5,077,211 of Yarosh, U.S. Pat. No. 4,621,023 ofRedziniak et al. or U.S. Pat. No. 4,508,703 of Redziniak et al. can beused. The compositions of the invention intended to target skinconditions can be administered before, during, or after exposure of theskin of the mammal to UV or agents causing oxidative damage. Othersuitable formulations can employ niosomes. Niosomes are lipid vesiclessimilar to liposomes, with membranes consisting largely of non-ioniclipids, some forms of which are effective for transporting compoundsacross the stratum corneum.

5. TREATMENT

The present invention also encompasses a method of treating a patientsuffering from a condition associated with PI-3 kinase activity. ThePI-3 kinase activity may be abnormal, excessive, or constitutivelyactive. The present invention also encompasses a method for treatinginflammatory disease comprising administering to a patient in needthereof a therapeutically effective amount of a compound of the presentinvention. Such diseases and adverse health effects attributable toinappropriate PI-3 kinase signaling activity have been disclosed in theart, for example U.S. 2002/0150954A1; U.S. Pat. No. 5,504,103; U.S. Pat.No. 6,518,277B1; U.S. Pat. No. 6,403,588; U.S. Pat. No. 6,482,623; U.S.Pat. No. 6,518,277; U.S. Pat. No. 6,667,300; U.S. 20030216389; U.S.20030195211; U.S. 20020037276 and U.S. Pat. No. 5,703,075 the contentsof which are incorporated by reference.

The present invention also encompasses a method for enhancing p53mediated programmed cell death comprising administering to a patient inneed thereof a therapeutically effective amount of a compound of thepresent invention.

The present invention also encompasses a method for enhancing thechemosensitivity of tumor cells comprising administering to a patient inneed thereof a therapeutically effective amount of a compound of thepresent invention.

The present invention also encompasses a method for enhancing theradiosensitivity of tumor cells comprising administering to a patient inneed thereof a therapeutically effective amount of a compound of thepresent invention.

The present invention also encompasses a method for inhibiting tumorinduced angiogenesis comprising administering to a patient in needthereof a therapeutically effective amount of a compound of the presentinvention.

The present invention also encompasses a method for inhibitingangiogenic processes associated with non-cancer diseases comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a compound of the present invention.

The present invention also encompasses a method for treatment of cancercomprising administering to a patient in need thereof a therapeuticallyeffective amount of a compound of the present invention.

The compound may be administered simultaneously or metronomically withother anti-cancer treatments such as chemotherapy and radiation therapy.The term “simultaneous” or “simultaneously” as used herein, means thatthe other anti-cancer treatment and the compound of the presentinvention administered within 48 hours, preferably 24 hours, morepreferably 12 hours, yet more preferably 6 hours, and most preferably 3hours or less, of each other. The term “metronomically” as used hereinmeans the administration of the compounds at times different from thechemotherapy and at certain frequency relative to repeat administrationand/or the chemotherapy regiment.

The chemotherapy treatment may comprise administration of a cytotoxicagent or cytostatic agent, or combination thereof. Cytotoxic agentsprevent cancer cells from multiplying by: (1) interfering with thecell's ability to replicate DNA and (2) inducing cell death and/orapoptosis in the cancer cells. Cytostatic agents act via modulating,interfering or inhibiting the processes of cellular signal transductionwhich regulate cell proliferation and sometimes at low continuouslevels.

Classes of compounds that may be used as cytotoxic agents include thefollowing: alkylating agents (including, without limitation, nitrogenmustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas andtriazenes): uracil mustard, chlormethine, cyclophosphamide (Cytoxan®),ifosfamide, melphalan, chlorambucil, pipobroman, triethylene-melamine,triethylenethiophosphoramine, busulfan, carmustine, lomustine,streptozocin, dacarbazine, and temozolomide; antimetabolites (including,without limitation, folic acid antagonists, pyrimidine analogs, purineanalogs and adenosine deaminase inhibitors): methotrexate,5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine,6-thioguanine, fludarabine phosphate, pentostatine, and gemcitabine;natural products and their derivatives (for example, vinca alkaloids,antitumor antibiotics, enzymes, lymphokines and epipodophyllotoxins):vinblastine, vincristine, vindesine, bleomycin, dactinomycin,daunorubicin, doxorubicin, epirubicin, idarubicin, ara-c, paclitaxel(paclitaxel is commercially available as Taxol®), mithramycin,deoxyco-formycin, mitomycin-c, 1-asparaginase, interferons (preferablyIFN-α), etoposide, and teniposide.

Other proliferative cytotoxic agents are navelbene, CPT-11, anastrazole,letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, anddroloxafine.

Microtubule affecting agents interfere with cellular mitosis and arewell known in the art for their cytotoxic activity. Microtubuleaffecting agents useful in the invention include, but are not limitedto, allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine(NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatin 10 (NSC376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel(Taxol®, NSC 125973), Taxol® derivatives (e.g., derivatives (e.g., NSC608832), thiocolchicine NSC 361792), trityl cysteine (NSC 83265),vinblastine sulfate (NSC 49842), vincristine sulfate (NSC 67574),natural and synthetic epothilones including but not limited toepothilone A, epothilone B, and discodermolide (see Service, (1996)Science, 274:2009) estramustine, nocodazole, MAP4, and the like.Examples of such agents are also described in Bulinski (1997) J. CellSci. 110:3055 3064; Panda (1997) Proc. Natl. Acad. Sci. USA94:10560-10564; Muhlradt (1997) Cancer Res. 57:3344-3346; Nicolaou(1997) Nature 387:268-272; Vasquez (1997) Mol. Biol. Cell. 8:973-985;and Panda (1996) J. Biol. Chem. 271:29807-29812.

Also suitable are cytotoxic agents such as epidophyllotoxin; anantineoplastic enzyme; a topoisomerase inhibitor; procarbazine;mitoxantrone; platinum coordination complexes such as cis-platin andcarboplatin; biological response modifiers; growth inhibitors;antihormonal therapeutic agents; leucovorin; tegafur; and haematopoieticgrowth factors.

Cytostatic agents that may be used include, but are not limited to,hormones and steroids (including synthetic analogs):17.alpha.-ethinylestradiol, diethylstilbestrol, testosterone,prednisone, fluoxymesterone, dromostanolone propionate, testolactone,megestrolacetate, methylprednisolone, methyl-testosterone, prednisolone,triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide,estramustine, medroxyprogesteroneacetate, leuprolide, flutamide,toremifene, zoladex.

Other cytostatic agents are antiangiogenics such as matrixmetalloproteinase inhibitors, and other VEGF inhibitors, such asanti-VEGF antibodies and small molecules such as ZD6474 and SU6668 arealso included. Anti-Her2 antibodies from Genetech may also be utilized.A suitable EGFR inhibitor is EKB-569 (an irreversible inhibitor). Alsoincluded are Imclone antibody C225 immunospecific for the EGFR, and srcinhibitors.

Also suitable for use as an cytostatic agent is Casodex® (bicalutamide,Astra Zeneca) which renders androgen-dependent carcinomasnon-proliferative. Yet another example of a cytostatic agent is theantiestrogen Tamoxifen® which inhibits the proliferation or growth ofestrogen dependent breast cancer. Inhibitors of the transduction ofcellular proliferative signals are cytostatic agents. Representativeexamples include epidermal growth factor inhibitors, Her-2 inhibitors,MEK-1 kinase inhibitors, MAPK kinase inhibitors, PI3 inhibitors, Srckinase inhibitors, and PDGF inhibitors.

A variety of cancers may be treated according to the present inventionincluding, but not limited to, the following: carcinoma including thatof the bladder (including accelerated and metastatic bladder cancer),breast, colon (including colorectal cancer), kidney, liver, lung(including small and non-small cell lung cancer and lungadenocarcinoma), ovary, prostate, testes, genitourinary tract, lymphaticsystem, rectum, larynx, pancreas (including exocrine pancreaticcarcinoma), esophagus, stomach, gall bladder, cervix, thyroid, and skin(including squamous cell carcinoma); hematopoietic tumors of lymphoidlineage including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkinslymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, histiocyticlymphoma, and Burketts lymphoma; hematopoietic tumors of myeloid lineageincluding acute and chronic myelogenous leukemias, myelodysplasticsyndrome, myeloid leukemia, and promyelocytic leukemia; tumors of thecentral and peripheral nervous system including astrocytoma,neuroblastoma, glioma, and schwannomas; tumors of mesenchymal originincluding fibrosarcoma, rhabdomyoscarcoma, and osteosarcoma; and othertumors including melanoma, xenoderma pigmentosum, keratoactanthoma,seminoma, thyroid follicular cancer, and teratocarcinoma.

Most preferably, the invention is used to treat accelerated ormetastatic cancers of the bladder, pancreatic cancer, prostate cancer,non-small cell lung cancer, colorectal cancer, and breast cancer.

The present invention also encompasses a method for treatingpancreatitis comprising administering to a patient in need thereof atherapeutically effective amount of a compound of the present invention.As discussed in Gukovsky et al., Gastroenterology, 126(2):554-66 (2004),inhibition of PI-3 kinase may prevent pancreatitis.

The present invention also encompasses a method for treating ulcerscomprising administering to a patient in need thereof a therapeuticallyeffective amount of a compound of the present invention. The presentinvention also encompasses a method for treating gastric cancer, such asstomach cancer, comprising administering to a patient in need thereof atherapeutically effective amount of a compound of the present invention.As discussed in Bacon et al., Digestive Disease Week Abstracts andItinerary Planner, Vol. 2003, Abstract No. M921 (2003) and Rokutan etal., Digestive Disease Week Abstracts and Itinerary Planner, Vol. 2003,Abstract No. 354 (2003), PI-3 kinase is involved in the adhesion ofHelicobacter pylori to gastric cells. Furthermore, Osaki et al., Journalof Cancer Research and Clinical Oncology, 130(1): 8-14 (2004) indicatesthat a PI-3 kinase inhibitor, such as LY294002, may be useful as ananti-tumor agent for gastric carcinoma.

The present invention also encompasses a method of improving theperformance of a stent comprising administering a therapeuticallyeffective amount of a compound of the present invention to a patientwith a stent, such as a cardiovascular stent. As discussed in Zhou etal., Arteriosclerosis Thrombosis and Vascular Biology, 23(11): 2015-2020(2003), inhibition of PI-3 kinase may prevent the “stretch” damage thataccompanies stent placement in vessels. The compounds of the presentinvention in the stent or polymer matrix thereof may improve solubilityin the stent coating matrix, improve aqueous/serum solubility, orimprove perfusion into the cells immediately adjacent to the stentplacement.

The present invention also encompasses a method for treating age-relatedmacular degeneration (AMD) comprising administering to a patient in needthereof a therapeutically effective amount of a compound of the presentinvention. As discussed in Retina, Feb. 18, 2004, inhibition of VEGFinhibits blood vessel overgrowth associated with AMD. The compounds ofthe present invention may treat AMD by inhibiting angiogenesis.

The present invention also encompasses a method for treatinghypertension comprising administering to a patient in need thereof atherapeutically effective amount of a compound of the present invention.As discussed in Northcott and Watts, Hypertension, 43(1): 125-130(2004), inhibition of PI-3 kinase may prevent the low extracellularconcentrations of Mg²⁺ that are associated with hypertension.

The present invention also encompasses a method for suppressingdifferentiation of progenitor cells, such as myeloid progenitor cells,comprising adding an effective amount of a compound of the presentinvention to progenitor cells. As discussed in Lewis et al.,Experimental Hematology, 32(1): 36-44 (2004), inhibition of the PI-3kinase pathway suppresses myeloid progenitor cell.

The present invention also encompasses a method for treating livercancer comprising administering to a patient in need thereof atherapeutically effective amount of a compound of the present invention.As discussed in Leng et al., Hepatology 38(4) Suppl 1: 401A (2003),LY294002 inhibits phosphorylation of Akt (serine/threonine proteinkinase B), which is an indicator in human liver tissues.

The present invention also encompasses a method for treating conditionsassociated with a mutant PTEN comprising administering to a patient inneed thereof a therapeutically effective amount of a compound of thepresent invention. PTEN is a tumor suppressor gene located on chromosome10q23 that has been identified in patients with Cowden disease. Asdiscussed in Vega et al., Journal of Investigative Dermatology, 121(6):1356-1359 (2003), mutant PTEN has reduced ability to inhibit theactivation of the proto-oncogene Akt. Inhibitors of PI-3 kinase mayinhibit phosphorylation of Akt, thereby reducing the effect of themutant PTEN.

a. Administration

Compositions of the present invention may be administered in any mannerincluding, but not limited to, orally, parenterally, sublingually,transdermally, rectally, transmucosally, topically, via inhalation, viabuccal administration, or combinations thereof. Parenteraladministration includes, but is not limited to, intravenous,intraarterial, intraperitoneal, subcutaneous, intramuscular,intrathecal, and intraarticular. The compositions of the presentinvention may also be administered in the form of an implant, whichallows slow release of the compositions as well as a slow controlledi.v. infusion.

b. Dosage

A therapeutically effective amount of the compound required for use intherapy varies with the nature of the condition being treated, thelength of time that activity is desired, and the age and the conditionof the patient, and is ultimately determined by the attendant physician.In general, however, doses employed for adult human treatment typicallyare in the range of 0.001 mg/kg to about 200 mg/kg per day. The dose maybe about 1 μg/kg to about 100 μg/kg per day. The desired dose may beconveniently administered in a single dose, or as multiple dosesadministered at appropriate intervals, for example as two, three, fouror more subdoses per day. Multiple doses often are desired, or required.

A number of factors may lead to the compounds of the present inventionbeing administered at a wide range of dosages. When given in combinationwith other therapeutics, the dosage of the compounds of the presentinvention may be given at relatively lower dosages. In addition, the useof targeting agents may allow the necessary dosage to be relatively low.Certain compounds of the present invention may be administered atrelatively high dosages due to factors including, but not limited to,low toxicity, high clearance, low rates of cleavage of the tertiaryamine. As a result, the dosage of a compound of the present inventionmay be from about 1 ng/kg to about 100 mg/kg. The dosage of a compoundof the present invention may be at any dosage including, but not limitedto, about 1 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 μg/kg, 125 μg/kg,150 μg/kg, 175 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275 μg/kg, 300μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 425 μg/kg, 450 μg/kg,475 μg/kg, 500 μg/kg, 525 μg/kg, 550 μg/kg, 575 μg/kg, 600 μg/kg, 625μg/kg, 650 μg/kg, 675 μg/kg, 700 μg/kg, 725 μg/kg, 750 μg/kg, 775 μg/kg,800 μg/kg, 825 μg/kg, 850 μg/kg, 875 μg/kg, 900 μg/kg, 925 μg/kg, 950μg/kg, 975 μg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70mg/kg, 80 mg/kg, 90 mg/kg, or 100 mg/kg.

The present invention has multiple aspects, illustrated by the followingnon-limiting examples.

Example 1 Preparation of LY294002

A 10 g sample of LY294002 was prepared according to Scheme 2 based onthe procedure described in Vlahos et al., J. Biol. Chem. 269(7): 5241(1994), the contents of which are incorporated by reference. Thedisplacement of the thiomethyl group of thoichromones such as 12 byamines has been described previously (Bantick et al., J. HeterocyclicChem, 18:679 (1981), the contents of which are incorporated byreference) as has the cyclization of methyl phenyl ketones such as 11with carbon disulfide with concomitant alkylation of the thiol anion(Vlahos et al. and Bantick et al.). Preparation of methyl ketones (e.g.,11) in a one-step reaction from the carboxylic acid (10) was performedusing the procedure described in Rubottom et al., J. Org. Chem., 48:1550(1983), the contents of which are incorporated by reference.

Example 2 Preparation of Quaternary Analogs of LY294002

Following the procedure of Scheme 3 the tertiary amine of LY294002 wasquaternized using iodomethane or benzyl chlorides under forcingconditions to yield compounds A052-10 and Compound 13B. Example 56describes the synthesis of methyl quaternary prodrug A052-10. Example 57describes the synthesis of a phthalimido quaternary prodrug A052-08.Example 58 describes the synthesis of a paracarboxy benzyl quaternaryA044-78. [0199][0215] describes the synthesis of a para-scn-benzylquaternary prodrug A044-80.

Example 3 Preparation of Chloromethyl Esters

Chloromethyl intermediates were prepared following the proceduredescribed in Tsujihara, Synth Commun, 24, 767, 1994. Briefly, theappropriate carboxylic acid was diluted in a 50/50 mixture ofdichloromethane/water. The mixture was cooled in an ice-water bath andsodium bicarbonate (4 equiv) and n-tetrabutyl ammonium hydrogen sulfate(0.05 equiv) was added. After stirring for 5 min, chloromethylchlorosulfate (1.1 equiv) was added. The solution was stirred vigorouslyovernight. The mixture was transferred to a separatory funnel with moredichloromethane and washed with saturated sodium chloride solution. Theorganics were dried over sodium sulfate and the solvent removed toprovide the product. The material was characterized by LC-MS and in somecases by 1H NMR spectroscopy. By this general procedure the followingrepresentative chloromethyl esters were prepared from the correspondingcarboxylic acids:

TABLE 1 RET SM RET STRUCTURE REF. NO. TIME* TIME**

A029-42 3.612 2.329

A029-44 4.273 3.327

A029-58 3.820 2.833

A029-60 4.077 2.956

A029-62 UD UD

A029-72 UD UD

A029-80 UD UD

A029-82 UD UD

A029-86 UD UD

A040-46 3.906 2.901

A040-58 UD UD *HPLC-MS retention time using UV detection; **HPLC-MSretention time of the starting carboxylic acid using UV detection; UD =undetectable due to lack of UV absorbance and no ionization by MS

Example 4 Conversion of LY294002 to Quaternary Prodrug

LY294002 (Compound 1) was dissolved in acetonitrile and then each of thechloromethyl esters (1-1.5 equiv) from Example 3 was added along with1-2 equivalents of sodium iodide. At room temperature, the reactionproceeded only slowly with the chloromethylesters to give very smallamounts of the quaternized amine product along with the precipitation ofsodium chloride. At 65° C., the reaction proceeded to completion usuallyin 4 hours. The reaction when complete (as judged by analysis by LC-MS)was filtered; concentrated and then purified on reverse phase HPLC. Thefractions were collected and lyophilized to give the desired products asfluffy powders. Examples prepared and purified in this manner are shownin the table below (the counter anions are not displayed but includedchloride, iodide, acetate or mixtures thereof).

TABLE 2 ELS REF YIELD STRUCTURE MW RET MS NO (mg) PURITY

494.6 2.718 M+ = 494 A041-49 400.6 93%

472.5 2.661 M+ = 472 A023-23 145 90%

5134.6 3.019 M+ = 513 A036-48B 194.1 97%

470.6 2.724 M+ = 470 A031-11 190.4 98.6%

484.6 2.926 M+ = 484 A031-14 204.8 98.5%

436.5 2.800 M+ = 436 A028-81 31 90%

451.5 2.082 M+ = 451 A029-92

380.4 2.204 M+ = 380 A040-70 264.0 97%

422 2.735 M+ = 422 A045-09 102.3 93.7%

Example 5 Halomethyl Ester Linkers

Based on the results of Example 3 and Example 4, halomethyl esterlinkers were prepared (Scheme 4 and chart). Compound B was prepared fromCompound A (commercially available) as described in Example 3. Thiscompound was converted into the more reactive iodomethyl ester (CompoundC) by a Finklestein reaction by dissolving in acetone or 2-butanone andthen dissolving 2-5 equivalents of sodium iodide whereupon the sodiumchloride precipitated and the iodomethyl ester (Compound C) was producedin solution. Compound C was isolated by stripping off the solvent anddissolving in a water immiscible solvent such as methylene chloride andextracting with water to remove the residual sodium iodide.

Compound E was prepared from Compound D (commercially available)Compounds F and G were prepared in a manner similar to the production ofCompounds B and C, respectively.

Example 6 Quaternization of LY294402 with Halomethyl Linkers

Halomethyl esters, including those of Example 5, were use to quaternizeLY294002 using conditions similar to the methodology in Example 4.Representative prodrugs comprising a linker with a free functional groupinclude the following:

Compound 1105 was prepared by mixing Compound 1101 with compound C inacetonitrile where both are soluble and the product Compound 1105precipitates out over a three day period and is washed with a smallamount of acetonitrile to give substantially pure Compound 1105(confirmed by LCMS).

Example 7 Preparation of Prodrugs Using Compound 1111

Compound 1111 was produced by the method shown in Scheme 5. Compound1110 was treated with neat trifluoroacetic acid for 1-3 hours and theTFA was blown off with argon and dried under vacuum to give a glassysolid comprised of Compound 1113. Compound 1113 was then dissolved in1-3 ml of thionyl chloride and heated at 65° C. for 3-8 hours. Thethionyl chloride was blown off with argon and then dried under highvacuum to give Compound 1111 in good yields as a glassy yellow solid.Compound 1111 can be reacted as a typical acid chloride with variousnitrogen-containing and hydroxyl-containing nucleophiles for example bysimply dissolving in methanol to give the corresponding methyl esterCompound 1112.

A sample of Compound 1111 was dissolved in acetonitrile and treated withat least 5 equivalents of different alcohols in separate vials. After 1hour the samples were analyzed by HPLC-MS and showed goodconversion >90% of Compound 1111 to the corresponding ester as shown andcharacterized in Table 3.

TABLE 3 Retention Structure of Ref Time MS+ Ester Formed No MW(minutes)* Found

A046-92-1 518.55 2.849 518

A046-92-2 492.55 2.788 492

A046-92-3 476.51 2.640 476

A046-92-4 528.59 2.970 528

A046-92-5 522.62 3.197 522

A046-92-6 524.55 2.490 524

A046-92-7 466.52 2.632 466

A046-92-8 480.54 2.787 480 *UV 214 nm

Example 8 Preparation of Protein Conjugate Prodrugs Using Compound 1111

Proteins are conjugated in largely aqueous solution (pH 7-9) (phosphatebuffer to carbonate buffer) using an excess a 2-10 fold excess ofCompound 1111 relative to the amino groups or hydroxyl groups to bemodified. The acid chloride Compound 1111 can be introduced in a mixedorganic-aqueous solution (such at 50/50 water/acetonitrile or 50/50water/THF) or stirred in methylene chloride in a two-phase reactionsystem at room temperature for 1-24 hours. Protein-conjugates can bepurified by dialysis or ultrafiltration and used directly.

A 500 μl aliquot of 5 mg/ml transferrin protein (Sigma) in 50 mM sodiumbicarbonate buffer was mixed with 100 up of 30 mM A024-79 (100 molarequivalents), which was prepared according to Example 12, in DMSO. After1 hour and 20 minutes of reacting at room temperature a 50 up sample wasremoved and passed through a Sephadex G-10 (700 molecular weight cutoff)column to separate protein from small molecules. An aliquot of thepurified conjugated protein eluent was then extracted with acetonitrileand no detectable Compound 1 was observed by LC-MS. The purifiedconjugated protein eluent was allowed to stand at room temperature 39hours at which time the protein mix was again extracted withacetonitrile and this time 15% of the maximum theoretical amount ofCompound 1 was detected. These results indicate a molar ratio of 15moles of prodrug were attached per mole of transferrin. These resultsdemonstrated the attachment of an electrophilic linker-bearing prodrugto a representative protein and demonstrated that over time asubstantial amount of a PI3 kinase inhibitor (compound 1) was releasedfrom the protein under aqueous conditions.

Example 9 Preparation of Resin-Bound Prodrugs Using Compound 1111

The peptide arg-gly-asp-ser (RGDS) was prepared on wang resin usingstandard FMOC/HOBT coupling peptide chemistry using all natural aminoacids. The resin-bound peptide was reacted with Compound 1111 in DMFfrom 1-24 hours, filtered and the resin washed with DMF and thenmethylene chloride and then treated with trifluoroacetic acid to cleavethe conjugate Compound 1126 from the resin (Scheme 6). Example 55describes a scaled up preparation of Compound 1111.

Example 10 Preparation of Prodrugs with Folate Targeting Agents UsingCompound 1111

Compound 1111 has an electrophilic group that may be reacted withnucleophilic amino groups under mildly basic organic or aqueousconditions (i.e. sodium bicarbonate buffer from 20 mM to 500 mM) to forma nonreversible thiourea link. Suitable nucleophilic amino groups arepresent on the targeting biomolecule folate. Folate molecules A and Cwere conjugated to Compound 1111 via an amino group in DMF by mixing inroughly equal proportions in the presence of the base triethylamine ordiisopropyl ethyl amine to yield compounds B and D (Scheme 7).

Example 11 Preparation of Prodrugs with Antibody Targeting Agents UsingCompound 1111

Compound 1111 is conjugated to monoclonal antibodies in aqueous pH 7 to9 and then separated by ultrafiltration or other standard methods ofseparating protein conjugates from small molecules. The conjugatedperformed can be prepared according to Example 8.

Example 12 Preparation of Prodrugs Using N-hydroxysuccinimide Esters

An ester less reactive than Compound 1111 was prepared by producing theN-hydroxysuccinimide active ester of Compound 1113 (Scheme 8). A 100 mgsample of Compound 1113 (A024-67) was dissolved in 1 ml of dry THF alongwith 53 mg of N-hydroxysuccinimide (2 equivalents). With stirring a 45up aliquot of 1 M dicyclohexycarbodiimide in methylene chloride (2equivalents) was added all at once. Within 3 minutes a heavy whiteprecipitate formed indicating the coupling reaction was occurring. Afterallowing the reaction to stir for 23 hours the reaction mix was filteredand the solvent removed from the filtrate to yield 172 mg of crudeactive ester product as a thick yellow oil, assigned Compound A024-79and showing a retention time of 2.334 minutes with the expected mass ofM+=535 found for this peak.

Using the same chemistry as described above for Compound 1111, A024-79was used to conjugate targeting proteins as described in Example 8 andused to conjugate to a polymer is described in Example 74.

Example 13 Preparation of Prodrugs Using Compound 1105

Compound 1105 has an electrophilic group that may be reacted withnucleophilic amino groups under mildly basic organic or aqueousconditions (i.e. sodium bicarbonate buffer from 20 mM to 500 mM) to forma nonreversible thiourea link. Suitable nucleophilic amino groups arepresent on targeting biomolecules such as peptides, proteins and smallmolecules bearing amine groups such as vitamin derivatives (A and C ofScheme 7). Representative examples of such products include Compounds Band D.

Example 14 Preparation of Derivatives of Morpholine Ring

The thiomethyl compound of Scheme 1 was prepared as described inExample 1. This compound was heated in an appropriate solvent with orwithout a catalytic amount of acetic acid with an excess of thenucleophilic amine compound until most of the thiomethyl compound wasconsumed. The mix was then subjected to preparative reverse phase LC-MSto isolate the desired morpholine analog. Compounds prepared in thismanner are shown in Table 4 along with their conditions of preparation,characterization and isolation shown in Table 5. The NMR data for thecompounds is shown in Table 6.

TABLE 4 Mol Yield Yield Structure Compound # Wt (mg) (%)

1153 352.42 32.6 24.8

1154 483.5 54.6 30.3

1155 335.4 24.7 19.8

1156 307.3  5.0 61.2  4.4 53.4

1157 305.4 100 (est) 87.7 (est))

1158 321.4 30   25.0

1159 295 62   56.4

1160 293.4 24.4 22.3

1161 295.3 71   64.5

1162 337.4  6.7 23.5

1163 337.4 57.7 45.9

1164 335.4 41.0 32.8

1165 265.31 17.2 17.2

1166 319.29  2.4  3.0

TABLE 5 Heat Reten- Com- Lot No. (° C.)/ tion pound (Prep Time TimeYield Yield No. Material) Solvent (min) Cat (min) (mg) (%) 1153 A037-36n-BuOH  115/350 no 3.407 32.6 24.8 1154 A036-08 n-BuOH  110/192 no 3.15654.6 30.3 1155 A037-19-3 n-BuOH 110/24 no 3.649 24.7 19.9 1156 A037-18-1n-BuOH 110/48 no 2.764 61.2 53.4 1157 A037-15 n-BuOH 110/24 no 3.837 10087 1158 A037-29 n-BuOH 110/4  no 2.753 30 25 1159 A037-31 n-BuOH 110/20no 2.753 62 56.4 1160 A037-48 EtOH  65/220 yes 3.666 24.4 22.3 1161A037-40 EtOH  65/48 yes 3.012 71 64.5 1162 A037-69 toluene  65/24 —3.410 28.5 23.5 1163 A037-99A n-BuOH  110/180 no 3.278 57.7 45.9 1164A037-99B n-BuOH  110/180 no 3.587 41 32.8 1165 A041-32 n-BuOH/ 110/24yes 3.247 17.2 17.2 DMF 1166 A041-25 n-BuOH  110/240 yes 3.427 11.1 9.3

TABLE 6 1153 ¹H NMR(CDCl₃): δ 1.60-1.65 (b s, 2H), 3.772-3.399 (t, 4H),3.506-3.533 (t, 4H), 5.474 (s, 1H), 7.367- 7.550 (m, 7H), 8.178-8.202(d, 1H, J = 7.9 Hz) 1154 ¹H NMR(CDCl₃): δ 3.533-3.644 (m, 4H) 5.491 (s,1H), 7.370-7.541 (m, 7H) 8.173-8.197 (d, 1H, J = 7.76 Hz) 1155 ¹HNMR(CDCl₃): δ 1.165-1.187 (d, 6H), 2.567-2.627 (t, 2H), 3.580-3.640 (m,4H) 5.496 (s, 1H), 7.389- 7.591 (m, 7H), 8.164-8.187 (d, 1H, J = 7.75Hz) 1157 ¹H NMR(CDCl₃): δ 1.573-1.698 (m, 6H), 3.344-3.370 (t, 4H),5.543 (s, 1H), 7.37-7.562 (m, 7H), 8.163- 8.186 (d, 1H, J = 7.8 Hz) 1158¹H NMR(CDCl₃): δ 1.498-1.547 (m, 7H), 1.873-1.997 (m, 3H), 2.600-3.200(b, 1H), 3.140-3.160 (m, 1H), 3.316-3.323 (m, 1H), 3.737-3.831 (m, 2H),4.011-4.037 (m, 1H), 5.624 (s, 1H), 7.387-7.582 (m, 7H), 8.163-8.187 (d,1H, J = 7.8 Hz) 1159 ¹H NMR(CDCl₃): δ 1.5-2.3 (b s, 2H), 3.015 (s, 3H),3.400-3.426 (t, 2H), 3.674-3.741 (t, 2H), 5.412 (s, 1H), 7.287-7.325 (t,1H), 7.404-7.489 (m, 6H), 8.066-8.090 (d, 1H, J = 7.75 Hz) 1160 ¹HNMR(CDCl₃): δ 1.077-1.178 (t, 6H), 3.255-3.308 (q, 4H), 5.447 (s, 1H),7.367-7.546 (m, 7H), 8.180- 8.204 (d, 1H, J = 8.01 Hz) 1161 ¹HNMR(CDCl₃): δ 3.326-3.358 (t, 2H), g 3.358 (s, 3H), g 3.517-3.542 (t,2H), g 5.099 (b s, 1H), g 5.427 (s, 1H), g 7.373-7.565 (m, 7H), g8.172-8.195 (d, 1H, J = 7.74 Hz) 1163 ¹H NMR(CDCl₃): δ 3.035 (s, 3H), g3.479-3.488 (d, 2H), g 3.806-3.888 (m, 4H), g 4.961-4.980 (t, 1H), g5.566 (s, 1H), g 7.385-7.489 (m, 4H), g 7.555-7.586 (m, 3H), g8.180-8.204 (d, 1H, J = 8.05 Hz) 1164 ¹H NMR(CDCl₃): δ 1.931-2.103 (m,4H), g 3.133-3.248 (b s, 5H), g 3.273-3.296 (m, 1H), g 3.368-3.394 (m,1H), g 3.990-3.999 (b s, 1H), g 5.403 (s, 1H), g 7.374-7.556 (m, 7H), g8.191-8.215 (d, 1H, J = 7.81 Hz)

Example 15 HPLC Analysis

HPLC analysis was performed on a Shimadzu LCMS-2010 and employed a flowrate of 3 ml/min and a starting B concentration of 5%. The B solvent waslinearly ramped to 95% concentration at 5.0 minutes, held at 95% until6.0 minutes, then linearly ramped back down to 5% at 6.5 minutes, whereit remains until the end of the run at 7.5 minutes. Unless otherwisenoted this is the method used in the examples. Method B is a slowgradient method for polar compounds that employs a flow rate of 3 ml/minand a starting B concentration of 0%, where it is held for the firstminute. The B solvent is linearly ramped to 10% concentration at 3.0minutes, then linearly ramped to 95% at 5.0 minutes, where it is helduntil 6.0 minutes, and then linearly ramped to 5% at 6.5 minutes, whereit remains until the end of the run at 7.5 minutes. In addition to massdetection the LC detection consisted of 3 channels; UV absorbance at 254nm, UV absorbance at 214 nm, and evaporative light scattering (AlltechELSD 2000). The evaporative light scattering detector was run at 50° C.with a nitrogen flow of 1.5 liters per minute. The CDL (chemicaldesolvation line) and block temperatures of the Shimadzu LCMS-2010 wereboth 300° C., and the nitrogen nebulizer gas flow was 4.5 L/min.Positive and negative mass spectra were detected from 50 to 2000 m/z.The column was a YMC CombiScreen ODS-AQ, S-5μ particle size, 50 mm longwith a 4.6 mm I.D. Mobile phase A was made using HPLC grade B&J waterwith 0.1% (v/v) HOAc added and mobile phase B was HPLC grade B&Jacetonitrile with 0.1% (v/v) HOAc added. This system gives a retentiontime of 1.50 to 1.60 minutes (t_(R)=1.50-1.60) for a standardcommercially available material (4-hydroxyphenylacetic acid; AldrichCatalog H5000-4; m.p. 149-151° C.) used as a reference standard.

Example 16 Preparative HPLC

Gradient Preparative HPLC was performed on a Shimadzu system composed oftwo LC-8A pumps connected to a SIL-10A autosampler and eluting over areverse phase column (YMC, cat CCAQSOSO520WT; ODS-AQ CombiPrep, 20 mm×50mm) and then passing through an MRA variable volume splitter; thesmaller stream was then made up to 3 ml/minute using a LC-10ADVP make-uppump (MeOH) and the eluent passed through a variable two channelwavelength UV detector and then split roughly 6:1 to an evaporativelight scattering detector (run at 50 C with a nitrogen flow of 1.5liters per minute) and a Shimadzu 2010 Mass detector; the larger streamfrom the MRA splitter then flowed to a Gilson 215 liquid handler servingas a fraction collector triggered by mass, UV absorbance, or ELS peaksize.

Different gradients were run always starting with the more aqueoussolvent A and ramping up to various concentrations of B. Mobile phase Awas made using HPLC grade B&J water with 0.1% (v/v) HOAc added andmobile phase B was HPLC grade B&J acetonitrile with 0.1% (v/v) HOAcadded.

Example 17 Bioactivity of Hydrolyzed Prodrugs

The bioactivity of LY294002 (Compound 1) was determined by assayingphagocytosis in J774 macrophages, which is a class I PI-3kinase-dependent pathway. Briefly, J774 cells are treated with LY294002at concentrations of 10, 1 and 0.1 μM along with an appropriate DMSOcontrol for 1 h in DMEM with 10% FCS and then sensitized sRBCs (sheepred blood cells) was added at a target to effector ratio equal to 100:1for 30 minutes at 37° C. Cells were exposed to hypotonic shock to removered blood cells and phagocytosis was determined by measurement ofhemoglobin concentration in the cell lysates. As shown in FIG. 1,LY294002 significantly blocked phagocytosis at all concentrations in adose dependent manner. These results indicate that the J774 cell systemcan be used to rapidly and easily assay the ability of the compounds ofthe present invention to inhibit PI-3 kinase activity. Using this methodof assaying PI-3 kinase activity targeted prodrug compound 1126 wastested at 5 μM concentration with variable preincubation time to allowfor in situ conversion of prodrug to active drug (compound 1). Thecontrol sample (time zero with no preincubation of compound 1126) showeda phagocytic index (PGI; a measure of the degree of phagocytosisoccurring as a result of not inhibiting PI-3 kinase) of 140 whereascompound 1126 with an aqueous pH=7 incubation time of 2, 5, and 10 hoursshowed PGI of 88, 78, and 37 respectively. This example demonstratesthat initially prodrug 1126 has little to no PI-3 kinase inhibitionactivity and over time is converted to bioactive drug that does showsignificant PI-3 kinase inhibition. Another experiment showed that a 20uM concentration of compound 1126 in an exposure-limited setting (20minute exposure to test solution then removal of test solution) had aPGI of 50 versus 163 for solvent blank, 190 for compound 1, and 170 forRGDS (tetrapeptide that is the targeting moiety of compound 1126). Thisexample showed the advantages of a targeted PI-3 kinase inhibitor in anexposure-time-limited setting. This effect was further evaluated byrepeating the experiment using decreasing doses of compound 1126 whichshowed that 10 uM, 3 uM, 1 uM, and 0 uM for a 20 minute incubation timefollowed by removal of the compound and then 2 hours incubation forphagocytosis to occur gave PGIs of 33, 143, 206, and 213 respectively.This example demonstrates the compound 1126 in a dose-exposure-limitedsetting inhibited PI-3 kinase in a dose dependent manner.

Example 18 Preparation of Bone Targeting Group A030-84

A solution of 500 mg 4-[(N—BOC)aminoethyl]aniline (Aldrich) in 10 mldioxane was treated with paraformaldehye (400 mol %, 270 mg) andtrimethylphosphite (400 mol %, 1.12 g). The mixture was heated to 95° C.overnight. Then more paraformaldehyde (270 mg) and trimethylphosphite(1.12 g) were added and it was heated at 95° C. overnight again. Thesolution was cooled, taken up in chloroform (20 mL) and washed withsaturated sodium chloride (20 mL) and water (20 mL). The organics weredried over sodium sulfate and the solvent and excess trimethylphosphiteremoved via rotary evaporation at 80° C. to provide 1.723 g of a clearoil. The presence of the title compound assigned lot number A030-74 wasconfirmed by electrospray HPLC-MS showing a retention time of t_(R)=2.9minutes and a mass of 467 m/z [M+H]+ and 489 m/z [M+Na]+ found for thedesired mass [M=C₁₈H₃₂N₂O₈P₂].

A solution of 870 mg of A030-74 as prepared above in 10 mLdichloromethane was treated with bromotrimethylsilane (690 mol %, 1.97g). The solution was stirred overnight. Methanol (10 mL) was added andthe solution was stirred 15 min and then concentrated to provide 1.12 gof an orange oil. The presence of the title compound was confirmed byelectrospray LC-MS. The retention time using this gradient was found tobe t_(R)=0.85 minutes and the mass spec for the desired product[M=C₉H₁₆N₂O₆P₂] found at the expected m/z 309 [M−H]⁻ operating in thenegative mode. This product was assigned reference number A030-84.

Example 19 Synthesis of Compound A014-52

To 1.0 g of 4-carboxyphenyl isothiocyanate was added dichloromethane (15mL) and distilled water (15 mL). The flask was cooled in an ice-waterbath and sodium bicarbonate (4.0 equiv) and n-tetrabutylammoniumhydrogen sulfate (0.05 equiv) were added. After 10 min, chloromethylchlorosulfate (1.2 equiv) was added. The solution was stirred vigorouslyovernight and transferred to a separatory funnel with the aid ofdichloromethane (10 mL). The layers were separated and the organic werewashed with saturated sodium chloride (20 mL). The organics were driedover sodium sulfate and the solvent removed to provide 1.10 g of a tansolid. The presence of the title compound was indicated by a shift inretention time in of the product (4.2 min) versus starting carboxylicacid (3.2 min). The compound was also confirmed by proton NMRspectroscopy: ¹H(CDCl₃) δ: 8.08 (d, 2H, J 8.8 Hz), 7.30 (d, 2H, J 8.8Hz), 5.95 (s, 2H).

Example 20 Synthesis of Compound A014-48

A solution of 250 mg of the chloromethyl ester (prepared via theprocedure described in A014-52) in 2 mL acetone was treated with sodiumiodide (1.2 equiv) and the solution was stirred overnight. The solutionwas filtered, the solvent removed, and the residue was taken up indichloromethane (10 mL). The solution was washed with 10% (w/v) sodiumsulfite (10 mL), 5% (w/v) sodium bicarbonate (10 mL), and water (10 mL).The organics were dried over sodium sulfate and the solvent was removedto provide 137 mg of a light green solid. The presence of the titlecompound was indicated by a shift in retention time in of the iodomethylester product (4.4 min) versus starting chloromethyl ester (4.2 min).The compound was also confirmed by proton NMR spectroscopy: ¹H(CDCl₃) δ:8.04 (d, 2H, J 8.8 Hz), 7.29 (d, 2H, J 8.1 Hz), 6.15 (s, 2H).

Example 21 Synthesis of Compound A014-76

A solution of 387 mg of the chloromethyl ester (prepared via theprocedure described in A014-52) in 6 mL 2-butanone was treated withsodium iodide (1.2 equiv) and the solution was heated 10 hr. Thesolution was filtered, the solvent removed, and the residue was taken upin dichloromethane (10 mL). The solution was washed with 10% (w/v)sodium sulfite (10 mL), 5% (w/v) sodium bicarbonate (10 mL), and water(5 mL). The organics were dried over sodium sulfate and the solvent wasremoved to provide 310 mg of a tan solid. The presence of the titlecompound was indicated by a shift in retention the iodomethyl esterproduct (4.4 min) versus starting chloromethyl ester (4.2 min).

Example 22 Synthesis of Compound A018-24

A solution of 64 mg of 2-p-nitrobenzyl-1,4,7,10-tetraazacyclododecane(Macrocyclics) in 500 μL dioxane was treated with paraformaldehyde (50mg) and trimethylphosphite (207 mg). The mixture was heated to 85° C.and then the solvent was removed via rotary evaporation at 75° C.Chloroform (10 mL) was added and the solution was washed with saturatedsodium chloride (2×10 mL) and water (2×10 mL). The organics were driedover sodium sulfate and the solvent removed to provide a brown oil. Thiswas purified via LC to provide the desired material. The presence of thetitle compound was confirmed by electrospray LC-MS A; t_(R)=1.8 min. MS[M=C₂₇H₅₃N₅O₁₄P₄] m/z 796 (MH⁺), 818 (MNa⁺).

Example 23 Synthesis of Compound A022-32

A solution of the 33 mg of the phosphonated macrocycle (prepared inA018-24) in 700 μL dichloromethane was treated with bromotrimethylsilane(72 mg). The mixture was stirred overnight and then morebromotrimethylsilane (36 mg) was added and it was stirred an additional3 days. Methanol (500 mL) was added and the solution stirred 1 hr, andthen the volatiles were removed to give a brown oil. Addition ofmethanol precipitated a brown solid, which was filtered and dried. Thiswas purified via LC to provide 2.7 mg of the desired material. Thepresence of the title compound was confirmed by electrospray LC-MS A;t_(R)=1.5 min. MS [M=C₁₉H₃₇N₅O₁₄P₄] m/z 682 (M−H⁻), 340 [(M−2H)/2)²⁻].

The nitro group is reduced using standard methods of reduction forexample stirring with 5% palladium on carbon catalyst in methanol underan atmosphere of pure hydrogen. The mixture is then filtered (takingcare to prevent air exposure to the catalyst) and the solvent evaporatedgive the amine.

Example 24 Synthesis of Compound A022-56

A mixture of phosphorus acid (1.26 g), 6 M hydrochloric acid (19.5 mL),and p-xylenediamine (1.0 g) was heated to 100° C. To this was added 37%(wt/wt) aqueous formaldehyde (1.15 mL) and the mixture was stirred at100° C. overnight. The mixture was filtered and the water removed byrotary evaporation at 80° C. to provide 2.11 g of a white solid. Thepresence of the title compound was confirmed by electrospray LC-MS usingmethod B; t_(R)=1.8 min. MS [M=C₁₀H₁₈N₂O₆P₂] m/z 325 (MH⁺).

Example 25 Synthesis of Compound A018-12

A solution of 928 mg N-BOC-1,4-diaminobutane in 10 mL dioxane wastreated with paraformaldehyde (592 mg) and trimethylphosphite (2.44 g).The mixture was stirred at 108° C. overnight and the solvent removed byrotary evaporation at 75° C. Chloroform (10 mL) was added and thesolution was washed with saturated sodium chloride (2×10 mL) and water(2×10 mL). The organics were dried over sodium sulfate and the solventremoved to provide 1.55 g of an oil. The presence of the title compoundwas confirmed by electrospray LC-MS; t_(R)=2.4 min. MS [M=C₁₅H₃₄N₂O₈P₂]m/z 455 (MNa⁺).

Example 26 Synthesis of Compound A026-92

A solution of 783 mg of the phosphonate (prepared in A018-12) in 18 mLdichloromethane was treated with bromotrimethylsilane (2.2 g). Thesolution was stirred overnight and methanol (10 mL) was added and themixture stirred for 2 hr. The volatiles were removed to provide 1.22 gof a yellow oil. The presence of the title compound was confirmed byelectrospray LC-MS using method B; t_(R)=0.4 min. MS [M=C₆H₁₈N₂O₆P₂] m/z275 (M−H⁻).

Example 27 Synthesis of Compound A030-74

A solution of 500 mg 4-[(N-BOC)aminoethyl]aniline in 10 mL dioxane wastreated with paraformaldehyde (270 mg) and trimethylphosphite (1.12 g).The mixture was heated to 95° C. overnight. Then more paraformaldehyde(270 mg) and trimethylphosphite (1.12 g) were added and it was heated at95° C. overnight again. The solution was cooled, taken up in chloroform(20 mL) and washed with saturated sodium chloride (20 mL) and water (20mL). The organics were dried over sodium sulfate and the solvent andexcess trimethylphosphite removed via rotary evaporation at 80° C. toprovide 1.72 g of a clear oil. The presence of the title compound wasconfirmed by electrospray LC-MS; t_(R)=2.9 min. MS [M=C₁₈H₃₂N₂O₈P₂] m/z467 (MH⁺); 489 (MNa⁺).

Example 28 Synthesis of Compound A030-84

A solution of 870 mg of the A030-74 in 10 mL dichloromethane was treatedwith bromotrimethylsilane (1.97 g). The solution was stirred overnight.Methanol (10 mL) was added and the solution was stirred 15 min and thenconcentrated to provide 1.12 g of an orange oil. The presence of thetitle compound was confirmed by electrospray LC-MS using method B;t_(R)=0.85 min. MS [M=C₉H₁₆N₂O₆P₂] M/Z found for 309 [M−H]⁻.

Example 29 Synthesis of Compound A035-66

A 2.0 g portion of 4-aminomethyl benzoic acid (Aldrich) was dissolved in20 mL of water containing 0.64 g of solid NaOH. A 3.18 g portion of Bocanhydride (Aldrich) was added and the mix allowed stir overnight. Themix was adjusted to pH=2 by the careful addition of 15 mL of 2N HCl. Theresulting white solid was filtered and dried to give 2.9997 g ofproduct. The product was characterized by LCMS (retention time 2.901minutes and desired M−H mass ion observed at 250 m/z).

Example 30 Synthesis of Compound A035-6

A 1.5 portion of A35-66 was dissolved in 17 mL of dry THF along with0.69 g of N-hydroxysuccinimide (Aldrich) and then treated all at oncewith 6 mL of 1M dicyclohexylcarbodiimide (Aldrich) in dichloromethanewith stirring. After two days the white precipitate (dicyclohexylurea)was filtered off and the filtrate rotoevaporated under vacuum to yield2.8146 g of white solid characterized by LCMS (retention time 3.299minutes and desired M+H observed at 349 m/z).

Example 31 Synthesis of Compound A035-14

A 500 mg portion of A035-6 was dissolved in 5 mL of dry THF and treatedwith 1.002 mL (10 equivalents) of ethylenediamine (EDA) and allowed tostir for 2 hours. The solution was then decanted from the formed solid.The solvent and excess EDA was then removed from the decanted solutionby rotoevaporation under vacuum to give 0.8728 g of white solid; theproduct was characterized by LCMS (retention time 1.608 minutes anddesired M+H observed at 294 m/z).

Example 32 Synthesis of Compound A032-24

A solution of 872 mg of the amine (prepared in A035-14) in 10 mL dioxanewas treated with paraformaldehyde (535 mg) and trimethylphosphite (2.21g). The mixture was heated at 100° C. overnight and then the solventremoved by rotary evaporation at 80° C. to give a brown solid.Chloroform (25 mL) was added and the solution was washed with water (15mL). The organics were dried over sodium sulfate and the solvent removedto provide 241 mg of a yellow semi-solid. This was purified via LC toprovide 58.8 mg of desired material. The presence of the title compoundwas confirmed by electrospray LC-MS; t_(R)=2.6 min. MS [M=C₂₁H₃₇N₃O₉P₂]m/z 538 (MH⁺), 560 (MNa⁺).

Example 33 Synthesis of Compound A032-40

A solution of 54.6 mg of phosphonate (prepared in A032-24) in 1 mLdichloromethane was treated with bromotrimethylsilane (156 mg). Themixture was stirred overnight. Ethanol (0.5 mL) and water (3 drops) wereadded and it was stirred 1 hr and then the volatiles were removed andthe material dried under vacuum. This was taken up in water (1 mL) andlyophilized to provide 59 mg of a tan solid. The presence of the titlecompound was confirmed by electrospray LC-MS using method B; t_(R)=0.4min. MS [M=C₁₂H₂₁N₃O₇P₂] m/z 380 (M−H⁻), 382 (MH⁺), 404 (MNa⁺).

Example 34 Synthesis of Compound A026-60

A026-60 prepared via the procedure of Kantoci, D., Kenike, J. K.,Wechter, W. J. Syn. Commun., 1996, 26(10), 2037: A mixture ofN-benzyl-N-methylamine (20.0 g), diethylphosphite (70.7 g) andtriethylorthoformate (29.3 g) was stirred under argon at reflux (150°C.) for 5 hr. Ethanol was removed via rotary evaporation at 70° C., andthe mixture was again heated at reflux overnight. The solution wasdiluted with 600 mL chloroform and washed with 1 M sodium hydroxide(3×100 mL) and saturated sodium chloride (3×150 mL). The organics weredried over sodium sulfate and the solvent removed to provide 74.0 g of alight yellow oil. 10.0 g of this material was subjected to silica gelcolumn chromatography using 14:4:1 ethyl acetate:hexane: methanol aseluent. This provided 6.08 g of a clear oil. The presence of the titlecompound was confirmed by electrospray LC-MS; t_(R)=3.4 min. MS[M=C₁₇H₃₁NO₆P₂] m/z 408 (MH⁺), 430 (MNa⁺), 471 (MNa—CH₃CN⁺).

Example 35 Synthesis of Compound A030-54

A030-54 (compound is known, CAS # 80475-00-9), prepared via theprocedure of Kantoci, D., Kenike, J. K., Wechter, W. J. Syn. Commun.,1996, 26(10), 2037: A solution of 4.52 g of the phosphonated benzylamine(prepared via A026-60) in methanol (45 mL) was treated with 10%palladium on carbon (200 mg) and subjected to an atmosphere of hydrogenovernight. The palladium/carbon was filtered to provide 2.98 g of alight yellow oil. The presence of the title compound was confirmed byelectrospray LC-MS using method A; t_(R)=1.9 min. MS [M=C₁₀H₂₅NO₆P₂] m/z318(MH⁺).

Example 36 Synthesis of Compound A039-16

A039-16: A 500 mg sample of 4-chloromethylbenzoic acid (Aldrich) wasdissolved in 8 mL of THF and treated all at once with 5 equivalents ofethylenediamine (Aldrich) (983 uL). After 24 hours the solvent wasstripped under high vacuum and the white solid (93%) was characterizedby LCMS (retention time 0.4 minutes and desired M+H observed at 195m/z).

Example 37 Synthesis of Compound A038-24

A mixture of 679 mg of the amino acid (prepared via A039-16), 37%(wt/wt) aqueous formaldehyde (1.04 mL), phosphorus acid (1.15 g), andconcentrated (12.1 M) hydrochloric acid (2.3 mL) in dioxane (10 mL) wasstirred at 100° C. overnight. The solvent was removed via rotaryevaporation at 75° C. and the mixture was centrifuged and the soliddiscarded. To the liquid was added more formaldehyde solution (1.04 mL),phosphorus acid (1.15 g), concentrated hydrochloric acid (2 mL) anddioxane (10 mL) and it was again was stirred overnight at 100° C. Thesolvent was removed via rotary evaporation at 75° C. to provide a thickoil. This was purified via LC to provide 276 mg of a brown solid. Thepresence of the title compound was confirmed by electrospray LC-MS usingmethod A; t_(R)=0.6 min. MS [M=C₁₃H₂₃N₂O₁₁P₃] m/z 475 (M−H⁻), 477 (MH⁺).

Example 38 Synthesis of Compound A038-50

A mixture of 6.0 g Fmoc-Lys-OH (Advanced ChemTech) in methanol (25 mL)and water (25 mL) was treated with 37% (wt/wt) aqueous formaldehyde(6.06 mL) and dimethylphosphite (8.96 g). The mixture was stirred at 80°C. for 2 hr, cooled, and extracted with dichloromethane (1×100 mL, 2×50mL). The organics were washed with saturated sodium chloride (50 mL),dried over magnesium sulfate for 30 min, and the solvent removed toprovide 10.17 g of a light green oil. The presence of the title compoundwas confirmed by electrospray LC-MS using method A; t_(R)=3.3 min. MS[M=C₂₇H₃₈N₂O₁₀P₂] m/z 613 (MH⁺), 636 (MNa⁺).

Example 39 Synthesis of Compound A038-66

A solution of 23.9 mg phosphonated Fmoc-Lys-OH (prepared via A038-50) indichloromethane (1 mL) was treated with bromotrimethylsilane (60 mg).The mixture was stirred overnight. The presence of the title compoundwas confirmed by electrospray LC-MS using method A; t_(R)=3.4 min. MS[M=C₂₃H₃₀N₂O₁₀P₂] m/z 555 (M−H⁻).

Example 40 Synthesis of Compound A038-76

A solution of 112.9 mg phosphonated Fmoc-Lys-OH (prepared via A038-50)in 6 M hydrochloric acid (3 mL) was stirred at 80° C. for 2 days. Water(9 mL) was added and after 2 more days the mixture was centrifuged andthe liquid decanted. The solid was dried under vacuum to provide 86.8 mgof an off-white solid. The presence of the title compound was confirmedby electrospray LC-MS using method A; t_(R)=3.1 min. MS[M=C₂₃H₃₀N₂O₁₀P₂] M/Z 555 (M−H⁻).

Example 41 Synthesis of Compound A038-90

A solution of 500 mg Fmoc-Lys-OH (Advanced ChemTech) in dioxane (5 mL)was treated with 37% (wt/wt) aqueous formaldehyde (303 μL), phosphorusacid (333 mg), and concentrated (12.1 M) hydrochloric acid (674 μL). Themixture was stirred at 90° C. overnight, the solvent was removed viarotary evaporation at 75° C. The presence of the title compound wasconfirmed by electrospray LC-MS using method B; t_(R)=5.4 min. MS[M=C₂₃H₃₀N₂O₁₀P₂] m/z 555 (M−H⁻).

Example 42 Synthesis of Compound A042-18

A solution of 1.29 g of the Boc-protected amino acid (prepared above aslot A035-66) in 15 mL tetrahydrofuran was treated withN-hydroxysuccinimde (623 mg) and 1.0 M 1,3-dicyclohexylcarbodiimide indichloromethane (5.4 mL). The mixture was stirred overnight and a whiteprecipitate was filtered and the supernatant concentrated to provide1.89 g of a white solid The presence of the title compound was indicatedby presence of a UV signal at 3.3 min.

Example 43 Synthesis of Compound A042-26

To a solution of 2.1 g tris-(2-aminoethyl)amine in 20 mL tetrahydrofuranwas added drop wise a solution of 1.0 g of the activated ester (preparedvia A042-18) in 20 mL tetrahydrofuran over a period of 40 minutes. Themixture was stirred overnight resulting in a precipitate that wasfiltered and concentrated via rotary evaporation to provide 2.10 g of ayellow oil. The presence of the title compound was confirmed byelectrospray LC-MS using method A; t_(R)=1.4 min. MS [M=C₁₉H₃₃N₅O₃] m/z380 (MH⁺), 402 (MNa⁺).

Example 44 Synthesis of Compound A042-32

A solution of 2.08 g amine (prepared via A042-26) in dioxane (20 mL) wastreated with paraformaldehyde (1.50 g) and dimethylphosphite (6.85 g).The mixture was stirred at 90° C. overnight and the solvent removed viarotary evaporation at 70° C. Dichloromethane (50 mL) was added and itwas washed with saturated sodium chloride (25 mL) and water (25 mL). Theorganics were dried over sodium sulfate and the solvent removed. Theresidue was purified via LC to provide 123.8 mg of a yellow oil. Thepresence of the title compound was confirmed by electrospray LC-MS usingmethod D; t_(R)=2.2 min. MS [M=C₃₁H₆₁N₅O₁₅P₄] m/z 868 (MH⁺).

Example 45 Synthesis of Compound A042-70

A solution of 111.1 mg of the phosphonated diamine (prepared viaA042-32) in 1 mL dichloromethane was treated with 194 mg ofbromotrimethylsilane. After 5 hr, methanol (1 mL) was added, the mixturewas stirred for 1 hr, and the solvent was removed to provide 113.9 mg ofa tan solid. The presence of the title compound was confirmed byelectrospray LC-MS using method B; t_(R)=1.0 min. MS [M=C₁₈H₃₇N₅O₁₃P₄]m/z 328 [(M+2H/2)²⁺)], 656 (MH⁺). The compound was also analyzed byproton NMR spectroscopy: ¹H(CDCl₃) δ: 7.77 (d, 2H, J 8.1 Hz), 7.43 (d,2H, J 8.2 Hz), 4.1-3.3 (m, 33H).

Example 46 Synthesis of Compound A026-94

A solution of 750 mg of the phosphonate (prepared via A030-54) in 24 mLdichloromethane was treated with bromotrimethylsilane (2.89 g). Thesolution was stirred overnight. Methanol (10 mL) was added and thesolution stirred for 2 hr and the solvent removed to provide a yellowoil which was lyophilized, resulting in 364 mg of a white solid. Thepresence of the title compound was confirmed by electrospray LC-MS usingmethod B; t_(R)=0.6 min. MS [M=C₂H₉NO₆P₂] m/z 204 (M−H⁻).

Example 47 Synthesis of Compound A042-96

A042-96 (tert-butylphosphite): A solution of 4.10 g phosphorus acid in100 mL tetrahydrofuran was treated with 2-methyl-2-propanol (7.41 g). A1.0 M solution of 1,3-dicyclohexylcarbodiimide in dichloromethane (100.0mL) was added, resulting in formation of a white solid. The mixture wasstirred overnight and the solid filtered and the solvent removed toprovide 6.82 g of a yellow oil. The presence of the title compound wasconfirmed by GC-MS. The following fragments were found: 57 [(CH₃)₃C⁺],83 [HP(OH)₃ ⁺], 123 [(HO)₂PC(CH₃)₂ ⁺].

Example 48 Synthesis of Compound A042-98

A mixture of ethanolamine (858 mg), paraformaldehyde (1.05 g),tert-butylphosphite (prepared via A042-96, 6.82 g) in benzene (100 mL)was heated at 90° C. overnight, resulting in a liquid atop a thick oil.The liquid was decanted and concentrated via rotary evaporation,resulting in an oil that was subjected to silica gel columnchromatography using 10% methanol in dichloromethane as eluant. A clearoil (436 mg) was obtained. The presence of the title compound wasconfirmed by electrospray LC-MS using method A; t_(R)=3.6 min. MS[M=C₂₀H₄₅NO₇P₂] m/z 496 (MNa⁺).

Example 49 Synthesis of Compound A029-34

In order to prepare 4-isothiocyanatophenyl acetic acid, 1.3 ml (13.5mmoles, 2 equivalents) thiophosgene was added to 1.0 g of 4-aminophenylacetic acid (6.61 mmoles) [Aldrich] and 3.73 g of anhydrous potassiumcarbonate (4 equivalents) suspended in 20 ml. dry THF. The suspensionwas stirred for 15 minutes at room temperature followed by a 4 hoursheating in an silicon oil bath at 85° C. The solution was cooled andpassed though a one inch celite bed in a filter syringe. The solutionwas collected in a round bottom flask and the solvent removed underreduced pressure.

The sample was stored in a vacuum dessicator for two hours thendissolved in acetone-water mixture, frozen and lypholized to give 1.65 gof a blackish solid. The compound was identified by a shift in retentiontime in the LCMS chromatogram to 3.32 minutes.

Example 50 Synthesis of Compound A040-22

In order to prepare 4-Isothiocyanatophenyl acetic acid chloromethylester, 0.530 g of 4-Isocyanatophenyl acetic acid (2.7 mmoles) wasdissolved in 5.0 ml methylene chloride in a glass vial, to which wasadded 0.044 g of tetra-n-butyl ammonium hydrogen sulfate (phase transfercatalyst—0.05 equivalents) and 0.866 g sodium bicarbonate (4equivalents) dissolved in 5.0 ml of water. The solution was stirred inan ice bath for ten minutes. To the cold mixture was added 0.520 g ofchloromethyl chloro sulfate (ACROS Chemical—1.2 equivalents) and stirredfor 4 hours with the temperature gradually coming to room temperature.The organic layer was separated in a separatory funnel, washed with 10ml saturated brine and dried over anhydrous sodium sulfate. The solventwas removed under vacuum to give 0.703 g of a crude oil. The product wasidentified by the formation of new peak in the LCMS with a retentiontime of 4.268 minutes and the disappearance of the peak corresponding tostarting material.

Example 51 Synthesis of Compound A040-26

In order to prepare the 4-isothiocyanato phenyl acetic acid quarternarysalt derivative of Compound 1101, Compound 1101 (0.300 g, 1.0 mmole) and0.582 g godium iodide (4 equivalents) were dissolved in 4.0 ml dryacetonitrile in a one dram vial. The 4-isothiocyanatophenyl acetic acidchloromethyl ester (compound A040-22) in 2.0 ml dry acetonitrile wasadded with stirring. The mixture was heated on a silicone oil bath at65° C. for five hours, monitoring the progress of the reaction by LCMS.When most of the Compound 1101 starting material was consumed thereaction mixture was purified using preparative LCMS retention time of3.019 minutes, m⁺=513. A yield of 194.1 mg of 94% pure product wasobtained and identified by LCMS. Compound A040-26 is then reacted withnucleophile-bearing targeting agents as described by the methodsdisclosed in the examples utilizing compound 1105 to prepare usefultargeted prodrugs.

Example 52 Synthesis of Compound A044-52

In order to prepare N-benzyl-N-methyl carbamoyl chloromethyl ester,0.300 mg (324 uL, d=0.942) benzylmethylamine and 640 uL diisopropylethyl amine (1.5 equivalents) were dissolved in 2.0 ml dry methylenechloride and cooled in an ice bath. When the solution was cooled, 330 uLchloromethyl chloroformate (1.5 equivalents) in 2.0 ml dry methylenechloride was added and stirred for 2 hours, gradually allowing thesolution to come to room temperature. The yellow solution was placed ina separatory funnel and washed with 2×10 ml 1N HCl, 1×10 ml water and2×10 ml 1 N sodium bicarbonate. The organic layer was dried over sodiumsulfate and concentrated under reduced pressure. The chloromethylcarbamate was obtained as 0.95 g of a yellow oil and identified in theLCMS as a uv active component with a retention time Of 3.520 minutes

In order to prepare the N-benzyl, N-methyl carbamoylmethyl quarternarysalt of Compound 1101, 0.5 g of Compound 1101 and 0.5 g sodium iodide(20 equivalents) were dissolved in 5.0 ml dry acetonitrile in a glassvial. The N-benzyl, N-methyl carbamoyl chloromethyl ester was added tothe solution then placed on an oil bath at 65° C. overnight. The productwas identified by LCMS and purified by preparative LCMS to give 169.5 mg(21.5% theoretical yield) of a solid assigned A044-52 with a retentiontime of 2.731 minutes, M+=485 and 90% purity.

Example 53 Synthesis of Compound A044-62

In order to prepare N-benzyl,N-methyl carbamoyl methyl quarternary saltof Compound 1157, 22 mg Compound 1157, 20 mg sodium iodide (2.0equivalents) and 30.7 mg N-benzyl-N-methyl carbonmoyl chloromethyl ester(2.0 equivalents) were mixed in 500 uL dry acetonitrile in a glass vial,and heated in an oil bath at 65° C. overnight. The product wasidentified via LCMS and purified by preparative LCMS. 5.4 mg wasobtained (15.5% theoretical yield) of a compound with a retention timeof 2.810 minutes, M+=483 and 98% purity.

Example 54 Synthesis of Compound A044-28

In order to prepare the benzyl formoyl-1-ethyl quarternary salt ofCompound 1101, 200 uL 1-chloroethyl chloroformate was added to 100 uLbenzyl alcohol in 2.0 ml dry methylene chloride in a glass vialincubated at 0° C. in an ice bath. To the cooled solution was added 200uL pyridine which caused formation of a white precipitate within a fewminutes. Stirring was continued at room temperature overnight. Ten ml ofmethylene chloride was added to the mixture that was then washed with1×10 ml 0.5 M HCl, 1×10 ml water and 1×10 ml 0.5 N sodium bicarbonatesolution. The organic layer was dried over sodium sulfate and thesolvent removed under reduced pressure. The benzyl-1-chloroethyl formatewas identified as a uv active component with a retention time of 3.933minutes.

To 100 mg Compound 1101 and 100 mg NaI (20 equivalents) dissolved in 1.0ml dry acetonitrile was added 107 mg (1.5 equivalents) of thebenzyl-1-chloroethyl formate. The mixture was heated at 65° C.overnight. The LCMS indicated the presence of starting material so anadditional 107 mg (1.5 equivalents) of benzyl-1-chloroether formate wasadded and the heating continued another 24 hours. The LCMS identified aproduct which could be separated from starting material with slowgradient chromatography. The desired compound was isolated usingpreparative LCMS to give 13.7 mg (8.7% theoretical yield) of a compoundwith a retention time of 4.690 minutes, M=+488 in 98.6% purity.

Example 55 Synthesis of Compound 1126

In order to prepare chloromethyl-t-butylsuccinate, mono t-butylsuccinate (Aldrich), 2.0 g, that was dissolved in 8.0 ml methylenechloride was added to a glass vial containing 4.0 g potassium carbonateand 0.24 g tetra n-butyl ammonium hydrogen sulfate in 8.0 ml water withstirring in an ice bath. After 15 minutes, 1.3 mL chloromethylchlorosulfate (Acros) was added to the methylene chloride layer and thereaction mixture was stirred with the temperature slowly coming to roomtemperature. The organic layer was separated and washed with 1×10 mlwater and 1×10 ml saturated brine solution. The solution was dried overanhydrous sodium sulfate and the solvent removed under reduced pressure.3 g of a pale yellow oil was obtained and assigned lot number A047-71.

The 3 g of A047-71 from above were dissolved in 36 mL of acetonitrileand treated with 1.8 g of Compound 1101 and 1.8 g of NaI and put on aheater with stirring for 16 hours. The reaction mix (includingprecipitate) was partitioned between water and methylene chloride andthe methylene chloride layer was separated, washed with brine, driedover sodium sulfate and evaporated to give a dark oil. This oil wasdissolved in 5 mL of acetonitrile and stored in the freezer for twodays. The yellow precipitate that formed was then filtered and washedwith 3 mL of acetonitrile and dried to give 2.8435 g of >95% purityproduct assigned lot A046-67A, retention time 2.719 minutes, [M+] of 494m/z. All of this product was dissolved in 10 mL of thionyl chloride andheated to 65° C. for 4 hours. The excess thionyl chloride was removedunder vacuum and the yellow oil dried under high vacuum to yield 1.9906g of the acid chloride (Compound 1111) as a yellow crunchy solid. Thissolid was used directly in subsequent reactions.

In order to couple the Compound 1101 acid chloride and de-FMOCed RGDSpeptide, 1.26 g acid chloride of Compound 1111 and 5.6 g de-FMOC removedRGDS peptide (both dried in vacuum dessicator over Phosphorouspentoxide) were mixed in a 50 ml round bottom flask under Argon gas. Tothe solids was added 270 uL dry pyridine in 28 ml methylene chloride andthe mixture shaken to dissolve the acid chloride. The mixture was placedon an orbital shaker for one hour. The solution was drained through afritted plastic syringe and washed with 2×10 ml methylene chloride andthe solvent drained. To the resin was added 500 uL anisole followed by20 ml of a 50/50 TFA/methylene chloride solution. The resin was allowedto stand in the TFA solution for three hours with occasional shaking.The TFA solution was drained away from the resin. The resin was washedwith 10 ml methylene chloride which was combined with the TFA solution.The TFA solution was placed into four vials that were blown dry withArgon gas. Each vial was treated with multiple ether washes and againblown dry with Argon gas. The product was identified by LCMS and waspurified in seventeen runs using preparative reverse phase LCMS. Thecombined runs yielded 163.7 mg of 96% pure product (assigned lota036-33) with a retention time of 1.768 minutes, M+=853 and [M+H]/2 at427 m/z. The LC-MS chromatogram and mass spectrum for this compound areshown in FIG. 7 and FIG. 8. In FIG. 7 the x-axis is time in minutes andthe y-axis for the top chromatogram is milli-absorbance units for the UVdetector at 254 nm and for the bottom chromatogram is millivoltsdetected by the evaporative light scattering detector. In FIG. 8 thex-axis is the mass-to-charge ratio (m/z) and the y-axis is the intensityof the mass ion count.

Example 56 Synthesis of Compound A052-10

In order to preparation the phthalimidomethyl quarternary salt ofCompound 1101, a mixture of 100 mg Compound 1101 and 100 mg sodiumiodide (2.0 equivalents) were dissolved in 3.0 ml dry acetonitrile. Tothe mixture was added 128 mg of chloromethylphthalimide (Aldrich) andthe vial was heated in an oil bath at 55° C. for four days. The productwas identified by LCMS as a new peak with a retention time of 3.984minutes, M+=467.

Example 57 Synthesis of Compound A052-08

In order to preparation the phthalimidomethyl quarternary salt ofCompound 1101, a mixture of 100 mg Compound 1101 and 100 mg sodiumiodide (2.0 equivalents) were dissolved in 3.0 ml dry acetonitrile. Tothe mixture was added 128 mg of chloromethylphthalimide and the vial washeated in an oil bath at 55° C. for four days. The product wasidentified by LCMS as a new peak with Rf=3.984, M+=467.

Example 58 Synthesis of Compound A044-78

In order to prepare the 4-Carboxybenzyl quarternary salt of Compound1101, 300 mg Compound 1101, 400 mg sodium iodide and 500 mg ofchloromethyl benzoic acid were mixed in a glass vial and suspended in4.0 ml dry acetonitrile. The reaction was heated on an oil bath at 65°C. and monitored for two weeks by LCMS. The solution was filteredthrough a fritted plastic syringe that had been fitted with anadditional 2 micron filter. The desired compound was isolated usingpreparative LCMC and had a retention time of 3.717 minutes, M+=442. Ayield of 27.7 mg of 96% purity was obtained. The carboxlic acid group ofcompound A044-78 is converted to a reactive group by a) reaction withN-hydroxysuccinimide as described by the method in Example 31 to preparethe NHS active ester or b) conversion to the acid chloride as describedby the method in Example 56. Either of these reactive groups are thenreacted with the nucleophilic amine or alcohol groups of targetingagents using methods described in the previous Examples to give targetedprodrug conjugates.

Example 59 Synthesis of Compound A044-80

In order to prepare the 4-Isocyanatobenzyl quarternary salt of Compound1101, a mixture of 300 mg Compound 1101, 450 mg sodium iodide (3.0equivalents) and 490 mg (3.0 equivalents) of 4-chloromethylbenzeneisocyanate was dissolved in 4.0 ml dry acetonitrile and heatedoil in an oil bath at 65° C. for 2 days. The LCMS indicated the reactionhad gone to completion because of the absence of starting material(Compound 1101). The product was identified as a new peak with aretention time of 4.577 minutes, M+=439. Compound A044-80 is reactedwith nucleophilic groups of various targeting agents to producecarbamate or urea linkages to the targeting agents by the methods ofExamples utilizing compound 1105 and A040-26.

Example 60 Synthesis of Compound A044-4

In order to prepare the pivaloylmethyl quarternary salt of Compound1101, 100 mg pivaloyl chloride (2.0 equivalents) was added dropwise to amixture of 100 mg Compound 1101 and 100 mg sodium iodide (2.0equivalents) in 2.0 ml dry acetonitrile. The mixture was heated on anoil bath at 65° C. for 2 hours. The solids were filtered using a frittedplastic syringe fitted with a 2 micron filter. The compound wasidentified and isolated using preparative LCMS. A yellow solid with aretention time of 2.735 minutes, M+=422 was obtained with a yield of102.3 mg of 93.7% purity.

Example 61 Synthesis of Compound A040-70

In order to prepare the acetoxymethyl quarternary salt of Compound 1101,1.0 g Compound 1101 was dissolved in 10 ml dry acetonitrile in a glassvial, and 1.0 g bromomethyl acetate (2.0 equivalents) was added andallowed to stir at room temperature overnight. LCMS indicated presenceof starting material, so the reaction was heated at 65° C. in an oilbath for 8 hours. The mother liquors were decanted from the solid andthe solids washed with a small amount of cold acetonitrile. The solidwas dried in a vacuum dessicator overnight. The product was identifiedas 264 mg of a white solid with a retention time of 2.204 minutes,M+=380 which was 97% pure. This compound was found to have very highwater solubility; a 32.5 millimolar solution of this compound inphosphate buffered saline was prepared by dissolving 27 mg in 1.865 mLof phosphate buffered saline with a resulting pH of around 4. A 50 uLaliquot of this solution (which contains 1.625 uMoles of Compound 1101as a prodrug) was injected in the tail vein of a nude mouse with noobservable untoward effects illustrating the lack of toxicity for theprodrug form whereas injection of 1.04 uMoles of Compound 1101 causedimmediate death in three out of three mice. Additionally, 250 uL of this32.5 mM solution of compound A040-70 was administered to a nude mouse byoral gavage. After 5, 15, and 30 minutes 40 uL of mouse blood wasobtained and analyzed by LC-MS (after extraction using acetonitrile) todemonstrate that combined blood levels of prodrug A040-70 and compound 1were 1.8, 4.97, and 4.80 micromolar at the respective time points. Thisresult demonstrates oral bioavailability of the active drug from theprodrug in vivo. The chloride salt is obtained by dissolving thiscompound A040-70 in a minimum amount of water and passing through ananion exchange resin bed such as Dowex 22 (chloride form) available fromAldrich and then washing with water and freeze-dry the combined eluantto give a solid possessing a chloride ion exchanged for the bromide ion.

Example 62 Preparation of a Polyoxyethylene-Bearing Prodrug for NonionicWater Solubility

A 78 mg portion of compound 1 was dissolved in 2 mL of acetonitrilealong with 76 mg of NaI and treated all at once with 144 mg ofchloromethylester A029-62 and stirred at 65° C. for 5 hours. Thereaction was purified by reverse phase LC-MS to give 72 mg (51% yield)of yellow solid A027-85 with a retention time of 2.30 minutes andcharacterized by having a mass spectrum showing M+=556 as expected forC30H38NO9. A small sample was introduced into phosphate buffer at pH=7.4followed by LC-MS over time whereupon a substantial portion of theprodrug converted back to compound 1 with an estimated half-life ofconversion of about 3 hours.

Example 63 Preparation of a Bone Targeted Prodrug of Compound 1

A 20 uL portion of 75 mMolar A042-70 (1.5 uMoles) in water was added toa vial containing 500 mMolar phosphate buffer (11 vials at 11 differentpHs ranging from 3.0 to 8.0 in 0.5 unit increments). After mixing eachvial was then treated with 50 uL of 60 uMolar Compound 1111 inacetonitrile (3.0 uMoles=2 equivalents relative to amino group of bonetargeting agent) and mixed by shaking. After one hour a 3 uL aliquot ofeach vial was injected on HPLC and the UV peak area determined forstarting material and desired product and by product of aqueoushydrolysis, Compound 1101. Only the samples at pH of 6, 6.5, 7, 7.5 and8 were found to have significant amounts of desired bone targetedprodrug A046-89P (retention time 2.50 minutes; [M+] found for 1075 m/zC42H59N6O19P4) [M+2]/2=538 m/z also found). This example demonstratesthat the optimum pH for synthesis of bone targeted prodrug under thesecondition is pH=7.0 which gave about 42% of theoretical yield of thedesired bone targeted prodrug of compound 1 possessing 4 phosphonic acidgroups. After 24 hours analysis of this same solution standing for thattime at pH=7.0 indicated that the targeted prodrug had convertedcompletely back to compound 1 demonstrating reversibility underphysiologically relevant conditions.

Example 64 In Vivo Efficacy of Compound 1126 Against Non-Small Cell LungCancer

Male nude mice of 4-6 weeks in age weighing around 30 grams wereinoculated subcutaneously in the right flank with 5 million tumor cells(human non-small cell lung cancer cells: H1299) on day 0. After 14 daysof allowing the tumors to grow the animals were divided into 3 groups of5 animals each. One group received vehicle control alone. One groupreceived twice-per-day tail vein injections (i.v) of 50 uL volume of24.4 millimolar solution of Compound 1126 in phosphate buffered salinecorresponding to 25 mg/kg/day dosing level of the active component ofthe prodrug (i.e. compound 1). The last group received twice-per-daytail vein injections (i.v) of 50 uL volume of 4.9 millimolar solution ofCompound 1126 in phosphate buffered saline corresponding to 5 mg/kg/daydosing level of the active component of the prodrug (compound 1). Thetumors were measured every three days using calipers to determine thetumor volume and the animals weights were recorded when the animals weresacrificed on day 27. The results are shown in Table 7 and indicatestrong tumor volume reduction versus control for both dose levels at thefirst datapoint only 3 days after treatment (Day 17) and continuingthrough to the end of the study:

TABLE 7 % Tumor Volume Reduction* % Tumor Volume Reduction* UsingCompound 1126 at Using Compound 1126 at 25 mg/kg/day 5 mg/kg/day Day 0 —— Day 17 35% 29% Day 20 66% 50% Day 24 68% 44% Day 27 68% 35% *Comparedto the vehicle only control animals

The twice per day doses were well tolerated over the two weekadministration period. The efficacy results above were also accompaniedby a lack of statistically significant difference in the animal bodyweights between the control group and the two treatment groups which asa general measure of gross toxicity indicated the targeted prodrug has adesirable lack of toxicity.

Example 65 In Vivo Efficacy of Compound 1126 Against Brain Cancer

An animal study was run as described in Example above except using ahuman brain cancer cell line (U87MG) and with treatment starting on day7 such that the first tumor volume measurement occurred on day 10. Theresults of Compound 1126 against this cancer cell line are shown inTable 8 and indicated effectiveness and a desirable lack of toxicity:

TABLE 8 % Tumor Volume Reduction* Using Compound 1126 at 25 mg/kg/day InU87MG xenograft model Day 0 — Day 10 20.2% Day 14 52.6% Day 17 38.5% Day21 35.2% *Compared to the vehicle only control animals

Example 66 Alpha v Targeted PI 3 Kinase Inhibitors Abrogated the TubeFormation of EDC-CBF1 Endothelial Cells on MATRIGEL

Tube formation represents to some extent the formation of angiogenesisin vivo. In this example it was determined to what degree PI 3 kinaseinhibitors (including targeted PI3 kinase inhibitor prodrugs) couldinhibit tube formation. Matrigel was plated into 12-well plate wells andsolidified in 37° C. for 2 hours. 1×10⁵ EDC-CBF1 endothelial cells werethen put on the top of the Matrigel layer in the presence of PBS, RADfV(cyclic negative control peptide), RGDfV (cyclic positive controlpeptide), RADS (linear negative control peptide), compound 1, orCompound 1126 at 20° M concentration overnight. Pictures were then takenusing a microscope. Well formed tubes can be visualized in the PBScontrol wells (top left panel of Figure). There was not much differencein the RGDfV-, RADfV-, or RGDS-containing wells compared with PBScontrol. Tube formation was significantly less in Compound 1101- andCompound 1126-containing wells.

Example 67 Targeted PI 3 Kinase Inhibitors Induced p53 TranscriptionalActivity in HBECs

This experiment tested the effect of PI 3 kinase inhibitors (Compound 1and the targeted prodrug version of Compound 1; Compound 1126) on theinduction of p53 luciferase activity. The transfection procedure wassimilar to that described in the literature to monitor p53transcription. Compound 1 (6 hour exposure) induced more than two foldhigher luciferase activity than the control and the targeted version ofcompound 1 (Compound 1126) had even better ability of inducing the p53luciferase activity (to almost 3 fold). This induction of p53 functionwas demonstrated to be abrogated by the p53 inhibitor, pifithrin alphaat 20 uM concentration. Co-transfection of catalytic active Akt alsoinhibited the p53 function induced by these compounds. This result showsthat the p53 transcription induced by PI3 kinase inhibitors isdownstream of Akt in the whole signaling cascade and the targetedprodrug Compound 1126 gave an enhanced induction of p53 versus theuntargeted drug, Compound 1.

Example 68 Purification of Compound 1126

Reaction mixture A044-84 (2.33 g) was weighed out into separate 0.33 gsamples and dissolved immediately before preparative chromatography in800 μl of a solution containing 1 part by volume acetonitrile, 1 part byvolume water, and 1% by volume acetic acid. 400 μl of this solution wasinjected for each preparative chromatography run. The pump A eluant wasB&J water (365-4) with 0.1% acetic acid added, and the pump B eluant wasB&J acetonitrile (015-4) with 0.1% acetic acid added. Initially, theeluant was 10% B, then linearly ramped to 34% B over a 4 minute period,then linearly ramped to 95% B at 4.25 minutes and held there until 5.25minutes, then linearly ramped back to the starting concentration of 10%B at 5.50 minutes. The total pump flow was 20 mL/minute.Re-equilibration of the system was accomplished while the autosamplerwas sampling for the next run. Using this gradient, the product withpositive mass spectral peaks at 853 (m/z=1) and 427 (m/z=2) eluted at3.37 minutes. Fractions were collected during the preparativechromatography runs when the signal detected at the ELSD exceeded 10 mv.Fractions containing product were diluted with a two-fold excess ofwater (by volume) and frozen in a lyophilization vessel using a dryice-acetone bath immediately after collection. After lyophilization overa 24-48 hour period a total of 180 mg of Compound 1126 as a white fluffysolid with a purity of 95% was obtained. This example demonstrates thatwith careful pH control the labile prodrug Compound 1126 can be isolatedin high purity using aqueous based reverse phase separation methods.

Example 69 Preparation of SCN-Reactive Prodrug

A 184 mg portion of A014-48 prepared by the method of Example 20 wasdissolved with 154 mg of Compound 1 in 12 mL of acetonitrile and stirredat room temperature. After 16 hours LC-MS indicated about 65% conversionto the desired quat compound so an addition 45 mg of the iodo compoundwas added and after an additional 22 hours the reaction had gone to 80%completion so an additional 40 mg of iodo compound was added and allowedto stir and additional 24 hours. The solids were then centrifuged andwash with acetonitrile to give 282.6 mg (45% yield) of the desiredproduct in high purity characterized by a retention time of 2.89 minutesand showing the desired M+ of 499 for C28H23N2O5S as compound COMPOUND1105.

Upon standing in methanol compound 1105, reacts cleanly with methanol togive compound A013-94 with a retention time of 2.78 minutes and theexpected M+ of 531 for C29H27N2O6S. This illustrates the reaction of theisothiocyanato prodrug with an alcohol to give a carbamate conjugationto the linker.

The isothiocyanate intermediate prodrug was also reacted with variousamines in methylene chloride (optionally in the presence oftriethylamine) including secondary amines to give urea products such asA017-55 (retention time 2.84 minutes showing the desired M+ of 644 m/zfor C35H38N3O7S); A017-57 (retention time 2.46 minutes showing thedesired M+ of 616 m/z for C33H34N3O7S); and A027-15 ((retention time2.82 minutes showing the desired M+ of 816 m/z for C38H48N3O11P2S. Theseexamples demonstrate reaction in good yield of the isothiocyanateprodrug with a diverse group of nucleophiles to give the desired linkedproducts. The A017-55 compound was further reacted with trifluoroaceticacid which surprisingly gave the cyclic compound A017-59 during thecourse of cleaving the t-butyl group (retention time 2.47 minutesshowing the desired M+ of 570 m/z for C31H28N3O6S). This exampledemonstrated additional chemistry can be performed on the linker groupof the prodrug compounds. In phosphate buffer at pH=7.4 significantamounts of this compound converted back to compound 1 over a 17 hourperiod, followed by LC-MS.

Example 70 Acute Toxicity of Compound 1 Versus Targeted Prodrug

Intravenously administered Compound 1 was determined to have a 100%mortality within 5 minutes of administration in three nude mice at adose level of 16 mg/kg. Compound 1126, prepared by the method of Example56, when injected intravenously at 37% higher dose level (22 mg/kg) inthree nude mice showed 0% mortality even after 1 hour of observation.This example demonstrates the improved formulation ability of theprodrugs and the diminished toxicity of the prodrugs of Compound 1.

Example 71 Analog of Compound 1

During the preparation of the intermediate A046-67SM such as describedfor Compound 1126 in Example 56 a side product was observed. Thismaterial was purified by LC-MS to give a single compound, A037-94,consistent by proton NMR with the proposed structure and giving aretention time of 3.00 minutes and a mass spectrum showing the expected[M+H]+ of 338 m/z (very low abundance) and more prominent mass[M+H+41(ACN)] at 379 m/z. This example demonstrates the isolation of anovel analog of compound 1 suitable for additional prodrug modification.

Example 72 Use of Prodrug to Deliver Compound 1 in Mice

Mice were injected with a million non-small cell lung cancer cells(H1299) subcutaneously and allowed to grow about 7 days until the tumormass was approximately 10 to 15 mm by 7 to 9 mm in dimensions. Animalswere injected with the targeted prodrug, Compound 1126, either i.v. (50uL) or i.p. (50 uL) with 32.6 mMolar solutions of Compound 1126 inphosphate buffered saline. After 60 minutes the mice were sacrificed andthe tumors removed. Three small pieces of the tumors were retrieved andminced. After aging for 24 hours to allow all of the prodrug to convertto compound 1 the tumor samples were extracted with acetonitrile.Quantitation by LC-MS indicated that the concentration of extractablecompound 1 (as the sum of free compound 1 and derived from Compound1126) was 157±7 nanomolar in the tumor pieces for the I.P. injection and271±17 nanomolar in the tumor pieces for the I.V. injection. Thisexample demonstrates the delivery of Compound 1 to tumor tissue using atargeted prodrug.

Example 73 Reversibility of Prodrugs to Form Compound 1

Prodrug Compounds were dissolved in water or in DMSO (if not freelysoluble in water) and then diluted at least 10-fold into 50 mM phosphatebuffer at pH=7.4 or pH=4.8 and allowed to stand at room temperature. Thefinal concentration of the compounds in aqueous environment ranged from50 to 500 uMolar. Aliquots over time were taken and analyzed by LC-MS todetermine both disappearance of prodrug and confirm appearance of drug(compound 1). Compound 1126 was found to have a half-life of about 1hour at pH=7.5 and a half-life of about 64 hours at pH=4.8. Compound1110 was found to have a half-life of about 10 hours at pH=7.4 andgreater than 120 hours at pH=4.8. Compound A040-70 was found to have ahalf-life of about 10 hours at pH=7.4. These examples demonstrate thatchemically the prodrugs converted to drug (compound 1) and thedisappearance of the prodrug is very pH dependant with conversion takingplace much faster at physiological pH and substantially slower at acidicpH.

Example 74 Synthesis of Tumor Localizing Conjugate

The electrophilic group-bearing compounds (such as compound A036-48B,1105, 1107, 1111, A024-79, and 1113) can be reacted with polymersbearing nucleophilic groups such as alcohols, amino, and thiol groups.N-(2-hyroxypropyl)methylacrylamide (HPMA) having molecular weight of2000 to 100,000 is reacted with excess compound 1111 in a nonproticorganic solvent such as methylene chloride or tetrahydrofuran in thepresence of triethyl amine or diisopropylethyl amine and then separatedby size exclusion chromatography, ultracentrifugation, or precipitationin another solvent such as methanol or ether. The polymer thusprecipitated or separated is substantially free of 1111 and is used as atumor localizing conjugate that releases active compound 1 overtime inthe vicinity of the tumor resulting in antitumor and anti-angiogeniceffects. Likewise polyglutamic acids can be converted topoly-nucleophilic bearing groups by reaction of the carboxylic acidswith excess diamines using carbodiimide coupling followed by sizeexclusion chromatography or reverse phase HPLC purification to obtainpoly-nucleophilic versions of polyglutamic acids. These polymers canthen be reacted directly with excess portions of compounds 1111 or 1105or A036-48B or A024-79 in an aprotic organic solvent such as methylenechloride or tetrahydrofuran in the presence of triethyl amine ordiisopropylethyl amine and then separated by size exclusionchromatography, ultracentrifugation, or precipitation in another solventsuch as methanol or ether. The poly-conjugated polymer thus precipitatedor separated is substantially free of low molecular weight residualprodrug and is used as a tumor localizing conjugate that releases activecompound 1 overtime in the vicinity of the tumor resulting in antitumorand anti-angiogenic effects.

1. A compound produced by a process comprising reacting Compound 2 ofthe formula:

with a halomethyl ester of the formula:

wherein, hal is a halogen atom; Z₁ and Z₂ represent O; Z₃ and Z₄represent O; R₁ and R₂ independently represent H, optionally substitutedaliphatic, optionally substituted aryl, hydroxyl, halogen, alkoxy,heterocycle, cyano, amino, or, are taken together to form an optionallysubstituted cycloaliphatic or optionally substituted aryl; R₃ representsH, optionally substituted aliphatic, and optionally substituted aryl; R₄and R₅ independently represent H, optionally substituted aliphatic,optionally substituted aryl, heterocycle, aryloxy, alkoxy, carboxy, or,are taken together to form an optionally substituted heterocycle oroptionally substituted heteroaryl; R₆ independently represent H,optionally substituted aliphatic, optionally substituted aryl,heterocycle, aryloxy, alkoxy, amino, or carboxy, and any of which areoptionally substituted with a targeting agent (T); and R₇ represents—CH₂—, —CH(CH₃), —CH(Ph), —C(CH₃)(COOH) or CH(CH(CH₃)₂.
 2. The compoundof claim 1, wherein R₁-Ring A-R₂ is selected from the group consistingof:

wherein R₄—N—R₅ is selected from the group consisting of:

wherein R₆ is selected from the givup consisting of:


3. The compound of claim 1, wherein R₆ is substituted with a targetingagent forming R₆-T, which is selected from the group consisting of:


4. The compound of claim 1, 2, or 3, wherein the targeting agent isselected from a carbohydrate, vitamin, peptide or peptidomimetic,protein, nucleoside, nucleotide, nucleic acid, liposome, lipid,bone-seeking agent, cartilage-seeking agent, diazepine, glucose,galactose, mannose or mannose-6-phosphate.
 5. The compound of claim 4,wherein the targeting agent is a peptide which is an RGD-moiety.
 6. Thecompound of claim 5, wherein the RGD-moiety is selected from the groupconsisting of RGDs, c(RGDfK), vitronectin, fibronectin,somatostatin-receptor agonists and somatostatin-receptor antagonists. 7.The compound of claim 1, wherein Compound 2 is of the formula:


8. The compound of claim 7, wherein R₆ is substituted with a targetingagent forming R₆-T, which is selected from the group consisting of:


9. The compound of claim 8, wherein the targeting agent is selected froma carbohydrate, vitamin, peptide or peptidomimetic, protein, nucleoside,nucleotide, nucleic acid, liposome, lipid, bone-seeking agent,cartilage-seeking agent, diazepine, glucose, galactose, mannose ormannose-6-phosphate.
 10. The compound of claim 9, wherein the targetingagent is a peptide which is an RGD-moiety.
 11. The compound of claim 10,wherein the RGD-moiety is selected from the group consisting of RGDs,c(RGDfK), vitronectin, fibronectin, somatostatin-receptor agonists andsomatostatin-receptor antagonists.
 12. The compound of claim 1, whereinthe halomethyl ester is halomethyl-t-butyl succinate.
 13. The compoundof claim 12, wherein the process further comprises: (a) removing thet-butyl group from the product produced by reacting Compound 2 withhalomethyl-t-butyl succinate, thereby producing a free carboxylic acidgroup; (b) converting the carboxylic acid group of the product of (a) toan acid chloride or an active ester; and (c) contacting the product of(b) with a targeting agent (I) comprising a free amino group.
 14. Thecompound of claim 13, wherein the halomethyl-t-butyl succinate ischloromethyl-t-butyl succinate, and wherein Compound 2 is LY294002. 15.The compound of claim 13, wherein the targeting agent (T) of step (c) isselected from a carbohydrate, vitamin, peptide or peptidomimetic,protein, nucleoside, nucleotide, nucleic acid, liposome, lipid,bone-seeking agent, cartilage-seeking agent, diazepine, glucose,galactose, mannose or mannose-6-phosphate.
 16. The compound of claim 15,wherein the targeting agent is a peptide which is an RGD-moiety.
 17. Thecompound of claim 16, wherein the RGD-moiety is selected from the groupconsisting of RGDs, c(RGDfK), vitronectin, fibronectin,somatostatin-receptor agonists and somatostatin-receptor antagonists.18. The compound of claim 16, wherein the protected RGDS-moiety has afree arginine alpha-amino group.
 19. The compound of claim 18, whereinthe process further comprises a step (d) removing a protecting groupfrom the RGDS-moiety.
 20. The compound of claim 19, wherein the processfurther comprises identifying and isolating the product of (d) usingpreparative reverse phase liquid chromatography mass spectrum (LCMS)wherein the isolated product has a mass of M+=853 in the positive modeof LCMS.
 21. The compound of claim 19, wherein the process furthercomprises exchanging any anion counterions with chloride counterions(salt).